Als2 gene and amyotrophic lateral sclerosis type 2

ABSTRACT

A human gene associated with amyotrophic lateral sclerosis type 2(ALS2) is provided. The gene is present in the second chromosome q33 region. Also provided are mutant versions of the gene as well as isolated nucleic acids derived from the gene and peptides encoded thereby. Methods for the diagnosis of ALS2 are alos provided.

RELATED APPLICATIONS

[0001] This application claims priority from U.S. application No.60/267,723 filed Feb. 12, 2001; Japanese application no. 2001-116973filed Apr. 16, 2001; and, U.S. application No. 60/318,352 filed Sep. 12,2001, which applications are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to genetic causes of amyotrophic lateralsclerosis of type 2 (“ALS2”).

BACKGROUND OF THE INVENTION

[0003] Amyotrophic lateral sclerosis (“ALS”) is a progressiveneurodegenerative disease in which distal and proximal motor neurons areselectively degenerated¹. Its cause is ambiguous and its onset is mostlyat middle age and thereafter. Its rate of onset is about 2-6 per 100,000persons and begins with lowering of muscular strength and myoatrophy ofwrist muscle as a secondary neuron hindrance resulting in bulbarparalysis symptoms such as atrophy of muscle of limbs, atrophy oftongue, alalia, dysphagia and dyspnea. No therapeutic method has beenestablished yet and most of the afflicted die within five years fromonset.

[0004] Juvenile amyotrophic lateral sclerosis of type 2 (“ALS2”;OMIM215100²) is a somatically recessive type hereditary disease.Although the frequency of its onset is rare, muscular convulsion oflimbs, face and throat is gradually expressed in persons of teens ortwenties and becomes chronic by bulbar paralysis as described above.

[0005] Amyotrophic lateral sclerosis of type 2 has been mapped to the1.7 cM interval flanked by D2S116 and D2S2237 on human chromosome2q33^(3,4). Alterations in 391 exons and their flanking regions derivedfrom 43 non-overlapping transcripts have been noted within thisinterval^(5,6).

[0006] ALS is a very severe disease and there is a need for developmentof means for its early detection or diagnosis and for treatment.

SUMMARY OF THE INVENTION

[0007] We have now identified a gene associated with amyotrophic lateralsclerosis type 2, termed the ALS2 or ALS2CR6 gene. This gene isexpressed in various human tissues including neurons in the brain andspinal cord, and encodes a protein with homology to RanGED and RhoGEF.

[0008] This invention now provides mammalian ALS2 genes and mutantversions thereof as well as peptides (including proteins) encoded bysuch genes. Also included are fragments and nucleic acids derived fromthese genes, corresponding peptides, and oligonucleotides suitable foruse as amplification primers and/or probes. Antibodies to the peptidesof this invention are also provided.

[0009] This invention also provides methods of diagnosis of ALS2 whichmay include identifying in a patient at risk, an altered ALS2 gene orprotein. The patient may be tested to characterize one or more mutationsin the gene or protein produced. Such a mutation may comprise theA261del mutation or the AG1548del mutations described herein.

[0010] This invention also provides nucleic acids which correspond to aregion of the ALS2 gene, which nucleic acids typically hybridize to atleast about 6, at least about 10, at least about 15, at least about 20,or at least about 25 consecutive nucleotides of an ALS2 sequence asdescribed herein, or to complements of such sequences, or to naturallyoccurring mutants or allelic variants thereof. The probes or primers maybe chosen to be capable of distinguishing (such as by amplification orhybridization) allelic variants, including the A261del and AG1548delmutations described herein. Such probes or primers may further include alabel which is capable of being detected. This invention also provideskits for identifying ALS2 genes, including those comprising allelesassociated with an ALS2 disease state, wherein the kits may comprise aprobe or primer as described herein. The kit may further compriseinstructions for using the probes or primers to distinguish alleles asdescribed herein.

[0011] This invention also provides vectors containing nucleic acids ofthis invention, including vectors adapted for expression of such nucleicacids in a target cell or organism. Such vectors may compriseappropriate transcription regulatory elements for directingtranscription of the nucleic acids in a target cell or organism. Nucleicacids and peptides of this invention may be expressed in bacterial aswell as eukaryotic cells, including mammalian cells. Such vectors may beadapted to express nucleic acids of this invention in a reversedirection so as to generate anti-sense transcription products.

[0012] This invention also provides non-human mammals comprising agenome in which an ALS2 gene has been mutated, including by deletion.Such a mammal may be a mouse and methods for altering the murine genomesuch as to produce an ALS2 “knock-out” mouse, are described herein andare known in the art.

[0013] This invention also provides the use of nucleic acids andpeptides as disclosed herein for the preparation of medicaments fortreatment of ALS2 or in the treatment of ALS2.

[0014] This invention also provides methods of treating patients forALS2, which methods may comprise testing the patient to diagnose orcharacterize an ALS2 disease state. A patient may be treated for ALS2,for example by administering to the patient or by otherwise providing anative form or functional fragment or derivative of the ALS2 peptidedescribed herein or such other therapeutic agent as which will restorefunction of the protein in a patient. Also included in this inventionare vectors suitable for use in gene therapy and gene therapymethodologies whereby a patient is treated to restore the function ofALS2 by delivering or producing a functional gene for expression in thepatient. Gene therapy vectors may, for example, be adeno-associatedvector, such as those known in the art. General methods for gene therapyare also known in the art.

[0015] This invention includes a human ALS2 gene which is present inhuman second chromosome q33 region and may code for a GTPase regulatoryfactor. The gene may encode an amino acid sequence of SEQ ID NO:2. cDNAsynthesized from mRNA that may be transcribed by this gene has a basesequence of SEQ ID NO:1.

[0016] This invention includes a human ALS2 mutated gene which isrelated to amyotrophic lateral sclerosis of type 2 and codes for amodified protein having an amino acid sequence of SEQ ID NO: 3 or SEQ IDNO:84, by a deficiency of one or two bases of the above human ALS2 gene.

[0017] This invention includes nucleic acids purified from genomic DNA,mRNA or cDNA as well as synthesized nucleic acids.

[0018] This invention includes oligonucleotides which hybridize to ALS2genes and variants thereof, preferably under stringent conditions.

[0019] This invention includes kits comprising oligonucleotides oroligonucleotide primer sets which may be used to carry out amplificationof ALS2 encoded nucleic acids, for example by the polymerase chainreaction (PCR).

[0020] This invention includes oligonucleotide probes which hybridize tothe regions containing base deficient sites (A261del and AG1548del) inALS2 under stringent conditions.

[0021] This invention includes oligonucleotide primer sets which carryout a PCR amplification of the region containing a base deficient sitein ALS2 as described herein. A specific example of this primer set is apair of synthetic oligonucleotides comprising the base sequences of SEQID NO: 6 and NO: 7 or a pair of synthetic oligonucleotides comprisingthe base sequences of SEQ ID NO: 8 and NO: 9.

[0022] This invention includes recombinant vectors comprising the abovenucleic acids and cells transcribed by said recombinant vectors.

[0023] This invention includes a GTPase regulatory factor or a GEF whichis characterized in being an expression product of an ALS2 gene asdescribed herein.

[0024] Embodiments of such GTPase regulatory factors of GEF's arerecombinant proteins produced by the transformed cells transformedaccording to this invention.

[0025] This invention includes a peptide comprising an amino acidsequence having continuous 5 or more acid amino residues in the first tothe 46th amino acid sequence in SEQ ID NO: 2 and also a peptidecomprising an amino acid sequence having continuous 5 or more acid aminoresidues in the 47th to the 1657th amino acid sequence in SEQ ID NO: 2.These peptides may be used for production of antibodies.

[0026] This invention also provides a modified protein which may be anexpression product of a mutant human ALS2 gene and which comprises theamino acid sequence of SEQ ID NO: 3. An embodiment of this modifiedprotein is a recombinant protein produced by a transformed cell.

[0027] This invention includes an antibody which recognizes peptides(including proteins) as disclosed herein. Embodiments of this antibodyare an antibody which is prepared using a peptide according to thisinvention as an antigen, including a peptide comprising an amino acidsequence having continuous 5 or more acid amino residues in the first tothe 46th amino acid sequence in SEQ ID NO: 2 and also an antibody whichis prepared using a peptide comprising an amino acid sequence havingcontinuous 5 or more acid amino residues in the 47th to the 1657th aminoacid sequence in SEQ ID NO: 2 as an antigen.

[0028] This invention furthermore provides methods for the diagnosis ofamyotrophic lateral sclerosis of type 2 which is characterized indetecting ALS2 mutated genes. An embodiment of this method for thediagnosis it that genomic DNA of the cells of a person to be diagnosedis subjected to a PCR amplification using a primer set comprising a pairof synthetic oligonucleotides comprising the base sequences of SEQ IDNO: 6 and NO: 7 or a pair of synthetic oligonucleotides comprising thebase sequences of SEQ ID NO: 8 and NO: 9, the resulting DNA fragmentsare treated with a restriction enzyme NarI and the said person whereeach of the DNA fragments is divided into two fragments is judged to besuffering from amyotrophic lateral sclerosis of type 2.

[0029] This invention also provides a method for the diagnosis ofamyotrophic lateral sclerosis of type 2 which is characterized in thatthe transcribed product of an ALS2 gene or mutated gene is detected. Inan embodiment of this diagnostic method, the transcribed product is cDNAor mRNA of the gene of an ALS2 mutated gene or the modified proteinexpressed by the said mutated gene. An embodiment of the case ofdetection of the modified protein is a method for the detection of theprotein where the antibody recognizing the first to the 46th amino acidsequences in SEQ ID NO: 2 reacts but the antibody recognizing the 47thto the 1657th amino acid sequence region in SEQ ID NO: 2 does not react.

[0030] Further, this invention provides a mouse ALS2 gene which may havean amino acid sequence of SEQ ID NO:5 as well as nucleic acids derivedtherefrom including nucleic acids synthesized or purified from genomicDNA, mRNA or cDNA of the mouse gene or a complementary sequence thereof.

[0031] This invention also provides a gene-defective non-human mammalsuch as a rodent, preferably a mouse, where function of an ALS2 gene issubstantially deficient. Also provided are tissues of such a mouse.

[0032] The human ALS2 gene according to this invention is a genomic genewhich has 33 introns and 34 exons, exists in a genomic DNA of 80.3 kbadjacent to a polymorphic DNA marker D2S2309 in human second chromosomeq 33 region (refer to FIG. 1) and codes for a human GTPase regulatoryfactor having an amino acid sequence of SEQ ID NO:2. In this ALS2 gene,its cDNA has a base sequence of SEQ ID NO: 1.

[0033] This invention provides an isolated nucleic acid that codes for apeptide having at least about 75, 80, 85, 90, 95, 97 or 100% identity toall of an amino acid sequence selected from the group consisting of: SEQID NO:2; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:84; and, amino acids372-1657 of SEQ ID NO:2. Also provided are the peptides encoded by thesenucleic acids.

[0034] This invention also provides an isolated nucleic acid consistingessentially of a nucleotide sequence having at least about 75, 80, 85,90, 95, 97 or 100% identity to all of a nucleotide sequence or acomplementary sequence thereof, selected from the group consisting of:SEQ ID NO:1; SEQ ID NO:4; nucleotides 124-5094 of SEQ ID NO:1;nucleotides 1225-5094 of SEQ ID NO:1; and, nucleotides 124-5076 of SEQID NO:4. Also provided are the peptides encoded by these nucleic acids.

[0035] The nucleic acids of this invention may be joined to a secondnucleic acid not naturally associated with the nucleic acid of thisinvention. By not naturally associated, it is meant that the secondnucleic acid is not part of an ALS2 gene and is not directly joined toan ALS2 gene in the genome of a mammal.

[0036] This invention also provides an oligonucleotide of 6 to 75nucleotides, wherein the oligonucleotide hybridizes to a nucleic acid ofthis invention or a complementary sequence thereof, under stringentconditions. An oligonucleotide of this invention may be joined to alabel, which is any moiety suitable for detectable labelling of thenucleic acid or for binding of the nucleic acid to a non-nucleic acidmoiety.

[0037] This invention also provides a peptide consisting essentially ofa sequence of at least 5 contiguous amino acids from a sequence selectedfrom the group consisting of: amino acids 1-46 of SEQ ID NO:2; aminoacids 47-1657 of SEQ ID NO:2; SEQ.ID NO:3; amino acids 43-49 of SEQ IDNO:3; SEQ ID NO:84; and amino acids 476 to 545 of SEQ ID NO:84. Thesepeptides are useful, for example in raising antibodies of this inventionand for investigating the function of the ALS2 protein.

[0038] This invention also provides a non-human mammal comprising amutated gene, wherein the gene but for the mutation would encode aprotein having at least about 75, 80, 85, 90, 95, 97 or 100% sequenceidentity to all of SEQ ID NO:2 or SEQ ID NO:5.

[0039] This invention also provides a method for the diagnosis ofamyotrophic lateral sclerosis type 2 in a patient, comprising detectingthe presence of a mutation in a gene that encodes a protein having atleast about 75, 80, 85, 90, 95, 97 or 100% sequence identity to SEQ IDNO:2 in a patient or a biological sample from a patient.

[0040] This invention also provides a method for the diagnosis ofanyotrophic lateral sclerosis type 2, comprising detecting the presenceor absence of a protein having at least about 75, 80, 85, 90, 95, 97 or100% sequence identity to all of SEQ ID NO:2 in a patient or abiological sample from a patient.

[0041] This invention also provides a method for the diagnosis ofamyotrophic lateral sclerosis type 2, comprising detecting the presenceor absence of a protein having at least about 75, 80, 85, 90, 95, 97 or100% sequence identity to all of SEQ ID NO:3 or SEQ ID NO:84 in apatient or a biological sample from a patient.

[0042] In the diagnostic methods of this invention, sequences may becompared to determine the presence of mutations; oligonucleotides may beused to detect hybridization to nucleic acids of the patient;amplification of nucleic acids of the patient may be performed; proteinsof the patient may be contacted with antibodies of this invention; orproteins produced in the patient may be evaluated for the function ofALS2 protein.

[0043] This invention also provides a method of treatment of amyotrophiclateral sclerosis type 2, comprising administering a peptide, a nucleicacid, or a pharmaceutical composition comprising the peptide or nucleicacid to a patient in need thereof, wherein the peptide comprises anamino acid sequence having at least about 75, 80, 85, 90, 95, 97 or 100%identity to SEQ ID NO:2 or a fragment thereof, and wherein the nucleicacid codes for said peptide.

[0044] This invention also provides a method of treatment of amyotrophiclateral sclerosis type 2, comprising administering a composition to apatient in need thereof, wherein the composition mimics the biologicalactivity of the peptide of SEQ ID NO. 2 or a fragment thereof.

[0045] This invention also provides the use of a peptide or a nucleicacid for preparation of a medicament for treatment of amyotrophiclateral sclerosis type 2, wherein the peptide comprises an amino acidsequence having at least about 75, 80, 85, 90, 95, 97 or 100% identityto SEQ ID NO:2 or a fragment thereof, and the nucleic acid codes forsaid peptide.

[0046] In this specification the term “isolated” with reference to anucleic acid or peptide means that a nucleic acid is separate from thegenome of a cell, a peptide is separate from a cell but does not meanthat the subject matter has been obtained from a genome or a cell. Insome instances, nucleic acids and peptides of this invention may besynthesized using conventional techniques.

[0047] Two nucleic acid or protein sequences are consideredsubstantially identical if, when optimally aligned, they share at leastabout 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 90% or at least 95%.Optimal alignment of sequences for comparisons of identity may beconducted using a variety of algorithms, such as the local homologyalgorithm of Smith and Waterman,1981, Adv. Appl. Math 2: 482, thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerisedimplementations of these algorithms (such as GAP, BESTFIT, FASTA andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, Madison, Wis., U.S.A.). Sequence alignment may also be carriedout using the BLAST algorithm, described in Altschul et al., 1990, J.Mol. Biol. 215:403-10 (using the published default settings).

[0048] Nucleic acid sequences of the invention may in some embodimentsbe substantially identical, such as substantially identical genetargeting substrates and target sequences. The substantial identity ofsuch sequences may be reflected in percentage of identity when optimallyaligned that may for example be greater than 50%, 80% to 100%, at least80%, at least 90% or at least 95%, which in the case of gene targetingsubstrates may refer to the identity of a portion of the gene targetingsubstrate with a portion of the target sequence, wherein the degree ofidentity may facilitate homologous pairing and recombination and/orrepair. An alternative indication that two nucleic acid sequences aresubstantially identical is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridization to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y.). Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point for thespecific sequence at a defined ionic strength and pH.

[0049] It is well known in the art that some modifications and changescan be made in the structure of a polypeptide without substantiallyaltering the biological function of that peptide, to obtain abiologically equivalent polypeptide. In one aspect of the invention, LPLS447X therapeutics may include peptides that differ from a portion ofthe wild-type LPL sequence by conservative amino acid substitutions. Asused herein, the term “conserved amino acid substitutions” refers to thesubstitution of one amino acid for another at a given location in thepeptide, where the substitution can be made without loss of function. Inmaking such changes, substitutions of like amino acid residues can bemade, for example, on the basis of relative similarity of side-chainsubstituents, for example, their size, charge, hydrophobicity,hydrophilicity, and the like, and such substitutions may be assayed fortheir effect on the function of the peptide by routine testing.

[0050] In some embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another having asimilar hydrophilicity value (e.g., within a value of plus or minus2.0), where the following hydrophilicity values are assigned to aminoacid residues (as detailed in U.S. Pat. No. 4,554,101, incorporatedherein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0);Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala(−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile(−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4).

[0051] In alternative embodiments, conserved amino acid substitutionsmay be made where an amino acid residue is substituted for anotherhaving a similar hydropathic index (e.g., within a value of plus orminus 2.0). In such embodiments, each amino acid residue may be assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe(+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser(−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln(−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

[0052] In alternative embodiments, conserved amino acid substitutionsmay be made where an amino acid residue is substituted for another inthe same class, where the amino acids are divided into non-polar,acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu,Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His;neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053]FIG. 1 is a transcription map of 3 Mb region of human chromosome2q33 including an ALS2 candidate region. The white open rectangle isbetween D2S116 to D2S2237^(2,3). Positions of 7 STS markers, 12polymorphic DNA markers and 42 independent transcription units areshown. Polarity of 38 transcription units are shown by arrows. Thelocation of the ALS2 gene is designated “ALS2CR6” which term may be usedinterchangeably for ALS2 below.

[0054]FIG. 2 shows a process for the detection of ALS2 associatedmutations. “a” is an example of the Tunisian and Kuwaiti ALS2 families.Genotypes of the members constituting a family is shown based onpreviously reported results^(3,4). “b” shows the result of sequencedetermination of mutation (A261del) in genomic DNA of the Tunisian ALS2family. Patient 10797 is A261del of a homozygotic type and the carrier10784 is a heterozygotic type. The sequence determination was carriedout for PCR products. “c” shows the results of determination of mutation(AG1548del) in the genomic DNA in the Kuwaiti ALS2 family. Sequence ofthe reverse strand of exon 5 in the region of interest are shown.Individual 18279 is a normal sibling, who is unaffected by ALS2 andcarries two normal haplotypes. The box in this sequence indicates theposition of the bases deleted in affected members. Individual 18281 isan unaffected parent who carries one disease haplotype. The overlappingnormal and mutated sequences are shown. Individual 18275 is affected andthe figure shows a homozygous CT deletion in the reverse strand of exon5. The position of the deleted bases is indicated by the arrow. Thecorresponding forward sequence and coded normal amino acids and novelamino acids produced by frameshifting are indicated. “d” showssegregation of the A261del mutation in the Tunisian ALS2 family. Thepresence of the deletion was assayed by the digestion with NarI, whichonly cuts mutated gene product. For exon-PCR products, the 339 bpfragment representing the normal allele was cleaved into two fragments(225 bp and 113 bp) in the mutant allele. For RT-PCR product, the 302 bpproduct which represents the normal allele was cleaved into twofragments (195 bp and 106 bp) in the mutant allele.

[0055]FIG. 3 shows northern blot analysis of the ALS2 (ALS2CR6) mRNA. In“a”, a northern blot containing 2 μg of poly A⁺ MRNA of many adult humantissues is hybridized with exon 4 of ALS2 cDNA. In the lower drawings,the same blot is hybridized with human β-actin cDNA for confirmation ofthe property and the comparative load of RNA. In the left, size of theALS2 transcript is shown. In “b”, northern blot containing 10 μg oftotal RNA obtained normal whole brain and 20 μg of total RNA obtainedfrom lymphocytes of patients and healthy persons (10788 persons) washybridized to exon 4 of the ALS2 cDNA. The right panel shows an agarosegel electrophoresis of an RNA sample.

[0056]FIG. 4 is a comparison of amino acid sequences in human ALS2CR6and mouse homolog mALS2CR6. The same residues are shown by frames. Thereare shown the position of the additional three amino acid residues ofthe Tunisian mutant protein (starting from the 47th amino acid residue),the position of the 25 amino acid residues (starting from the 372ndresidue) of a short variant part of the ALS2 gene and the position ofthe additional 70 amino acid residues of the Kuwaiti mutant protein(starting from the 476^(th) residue).

[0057]FIG. 5 shows an expression of ALS2 MRNA in brain and spinal cordof adult mouse. “a” is an arrow-like whole image of an RNA/RNA in situhybridization using an antisense ALS2 riboprobe while “b” is a controlimage using a sense strand probe. Significant expression was noted inneurons of hippocampus and dentate gyrus (c and g), Purkinje cells ofcerebellum (d and h), neurons of cerebral cortex (e and i) and cinereaof spinal cord including anterior horn cells (f and j). A scale barshows a length of 10 μm.

[0058]FIG. 6 is a result of an amino acid sequence analysis. “a” is aschematic chart of domains and motifs in normal and mutated ALS2protein. RCC1 is a regulatory factor for chromosome condensation, DH isa homologous domain to Db1, PH is a pleckstrin-homologous domain, MORNis membrane structure and recognition nexus and VPS9 is a vacuoleprotein for discrimination of 9 domains. “b” is comparison of amino acidsequences of RCC1 repeat-containing regions for human ALS2 (hALS2CR6),mouse ALS2 (mALS2CR6), human (h) RCC1, human (h) RPGR and mouse (m)RPGR. The amino acid residues shown by open frames are the same.Conserved amino acid residues are abundantly contained as well.Positions of the seven blades corresponding to RCC1 are shown accordingto the literature³⁰.

[0059]FIG. 7 is a chart that compares the wild type human, mouse, andshort human variant of the ALS2 proteins and the coding products of theA261del (Tunisian) and AG1548del (Kuwaiti) mutations.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The locus of a 1.7 cM region specified by microsatellite markersD2S116 and D2S2237 of a human second chromosome q 33 region has beenmapped^(3,4). The inventors previously prepared a physical map on thebasis of YAC/BAC/PAC of 3Mb genomic region covering the candidate regionin FIG. 1^(5,6). Sequences of cDNA clones and EST's have now beenanalyzed and 42 non-duplicated transcription units including 10 newgenes mapped. 411 pairs in of primers were designed depending upongenomic DNA of 14 persons of a family of ALS2 (FIG. 2a) and 6 normalcontrol persons having no kinship with the former was amplified by PCR.Seventy-seven base sequence polymorphs of introns or exons wereidentified by determining the sequence for all of the PCR products.Among them, a gene having base deletions related to onset of ALS2 wasidentified.

[0061] The ALS2 gene also includes restriction regions and regulatoryregions (promoter/enhancer, suppressor, etc.) which function inexpression of protein which is coded thereby. Such restriction andregulation regions are useful for clarifying the functions of the ALS2gene product as a GEF or a GTPase regulatory factor.

[0062] This ALS2 gene may, for example, by isolated by screening a humangenome library using pure polynucleotide or oligonucleotide comprising abase sequence of SEQ ID NO: 1 or a partial sequence thereof as a probe.The resulting genomic gene may be amplified by commonly used geneticamplifying methods such as, for example, a PCR (polymerase chainreaction) method, an NASBN (nucleic acid sequence based amplification)method, a TMA (transcription-mediated amplification) method or an SDA(strand displacement amplification) method.

[0063] A pure polynucleotide (DNA fragments and RNA fragments) may alsobe prepared from this ALS2 genomic gene, mRNA transcribed by this geneor cDNA synthesized from mRNA. For example, cDNA may be synthesizedusing poly(A)+RNA extracted from human cells as a template. The humancells may be either those excised from human body by operation, etc. orincubated cells. cDNA may be synthesized by known methods (Mol. CellBiol., 2, 161-170, 1982; J. Gene, 25, 263-269, 1983; Gene, 150, 243-250,1994). One may also synthesize cDNA by an RT-PCR method using anoligonucleotide as a primer and mRNA isolated from human cells as atemplate. Specifically, the cDNA prepared as such has a base sequence ofSEQ ID NO: 1. Those polynucleotides may be used for recombinantexpression of a human GTPase regulatory factor.

[0064] The oligonucleotides of this invention are DNA fragments or RNAfragments which hybridize to the above-mentioned ALS2 or theabove-mentioned nucleic acids under stringent conditions. For example itis a continuous DNA fragment of 10-100 bp in the base sequence of SEQ IDNO: 1. Here, stringent conditions means a condition whereby a specifichybrid formation of target with a probe is made possible by saltconcentration, concentration of organic solvent (such as formamide), ortemperature condition during hybridization and washing steps. Methodsare described in U.S. Pat. No. 6,100,037.

[0065] One methodology for creating stringent hybridization conditionsis [insert B & K)

[0066] A primer set of this invention is typically a pair ofoligonucleotides for amplification of ALS2 gene or related nucleicacids. Such a primer set may be designed on the basis of the basesequence of SEQ ID NO: 1, synthesized and subjected to purificationusing known methods. Size (base numbers) of the primer preferably is15-40 bases or more preferably, 15-30 bases which specificity annealwith a template DNA. However, when LA (long accurate) PCR is carriedout, it is effective to use primers in excess of 30 bases. A pair (two)primers comprising sense strand (5′-terminal side) and antisense strand(3′-terminal side) should not be complementary. In addition, aself-complementary sequence is to be avoided in a primer to prevent theformation of a hairpin structure. Further, in order to ensure a stablebond to a template DNA, the GC content should be about 50% andoccurrence of GC-rich or AT-rich regions in a primer should be avoided.Since an annealing temperature is dependent upon Tm (meltingtemperature), primers having Tm of 55-65° C. are chosen so as to preparea PCR product having a high specificity. The final concentration of theprimer used in PCR should be about 0.1 to about 1 μM. It is possible touse commercially available software for designing a primer including theOligo™ software [manufactured by National Bioscience Inc. (U.S.A.)] andGenetyx™ software [manufactured by Software Development KK (Japan)].

[0067] Mutated ALS2 genes may be obtained by a method where a DNAlibrary prepared from cells of a patient thought to be suffering fromALS2 is screened with a probe which hybridizes to a region containingmutant (e.g. a base deficient site) under a stringent condition. Purepolynucleotide (DNA fragment or RNA fragment) may be obtained fromgenomic DNA, mRNA or cDNA of an ALS2 mutated gene or a complementarysequence thereof. For example, an ALS2 mutated gene comprises a nucleicacid where the 261st base a of SEQ ID NO: 1 is deficient. Such apolynucleotide may be used for recombinant production of ALS2 modifiedprotein or for diagnosis of ALS2.

[0068] A primer set for a PCR amplification of ALS2, including variousregions having base deficient sites in mutated ALS2 is (for example) apair of synthetic oligonucleotides comprising base sequences of SEQ IDNO: 6 and NO: 7. This primer set is capable of a PCR amplification ofthe region (339 bp) including exon 3 and introns before and after thatin the ALS2 gene. Another primer set may be composed of syntheticoligonucleotides comprising base sequences of SEQ ID NO: 8 and NO: 9 andis capable of PCR amplification of exons 2-4 (302 bp) of the ALS2 geneusing RNA as a template. Any PCR product not cleaved by the restrictionenzyme NarI is derived from the normal ALS2 gene but PCR productsderived from a mutated ALS2 gene may be cleaved by NarI to give twofragments (FIG. 2c).

[0069] A recombinant vector of this invention may be a cloning vector oran expression vector. Vectors will be constructed depending upon thetype of the polynucleotide as an insert or upon the object for use. Forexample, when an ALS2 protein or a modified protein thereof is producedusing cDNA or its ORF region as an insert, there may be used anexpression vector for an in vitro transcription or an expressionsuitable for each of prokaryotic cells such as Escherichia coli andBacillus subtilis and eukaryotic cells such as yeast, insect cells andmammalian cells. When a genomic DNA of the ALS2 gene or a mutated genethereof is used as an insert, it is also possible to use a BAC(bacterial artificial chromosome) vector or a cosmid vector. Suchrecombinant vectors are also useful, for example, as probes fordiagnosis of chromosome abnormality by hybridization includingfluorescent in situ hybridization (FISH). Further, a nucleic acidderived from a normal ALS2 gene may be recombined in a virus vector suchas adenovirus or the like and the product may be used for genetictherapy.

[0070] In the manufacture of ALS2 peptide (including protein), atransformed cell of this invention may be a prokaryotic cell such asEscherichia coli and Bacillus subtilis or an eukaryotic cell such asfrom yeast, insects, and mammals. In addition, cells (such as blood stemcells) derived from a patient suffering from ALS2 which are transformedby a virus vector of this invention in which a nucleic acid derived froma normal ALS2 gene is recombined, may be used for a genetic therapy ofALS2. Such transformed cells may be prepared by introducing arecombinant vector into cells by means of known methods such aselectroporation, calcium phosphate method, liposome method and DEAEdextran method.

[0071] A peptide of this invention may be an expression product of anormal ALS2 gene or an expression product of a mutated ALS2 gene. Thenormal gene product is a GTPase transcription factor or GEF having anamino acid sequence of SEQ ID NO: 2. Peptides of this invention areuseful as immunogens for the preparation of an antibody, as targetmolecules for the development of therapeutic agents for ALS2, etc. Thesepeptides may be prepared by methods involving isolating peptides fromthe cells of healthy persons or patients suffering from ALS2. Methods ofchemical synthesis on the basis of a desired amino acid sequence fromSEQ ID NO:2 or SEQ ID NO:3, etc. and (preferably) by production andisolation or purification from the above-mentioned transformed cells.Such transformed cells are incubated and isolation and purification arecarried out for the culture by, for example, means of treatment with amodifier such as urea or with a surface-active agent, ultrasonic wavetreatment, enzymatic digestion, precipitation by salting out or bysolvent, dialysis, centrifugal separation, ultrafiltration, gelfiltration, SDS-PAGE, isoelectric electrophoresis, ion exchangechromatography, hydrophobic chromatography, affinity chromatography andreversed phase chromatography. Such proteins may include fused proteinswith any other protein. For example, fused proteins withglutathione-S-transferase (GST) or green fluorescent protein (GFP) maybe exemplified. In addition, the protein expressed in cells may besubjected to various kinds of modifications in the cells after beingtranslated. Accordingly, modified proteins are also included in thecoverage of the protein of this invention. Examples of the modificationafter translation as such are elimination of N-terminal methionine,N-terminal acetylation, addition of sugar chain, limited decompositionby intracellular protease, myristoylation, isoprenylation andphosphorylation.

[0072] An antibody of this invention is a polyclonal antibody ormonoclonal antibody which recognizes a peptide of this invention.Examples include an antibody prepared using a peptide comprising anamino acid sequence of continuous 5 amino acid residues or more of thefirst to the 46th amino acid sequence in SEQ ID NO: 2 as an antigen andan antibody prepared using a peptide comprising an amino acid sequenceof continuous 5 amino acid residues or more of the 47th to the 1657thamino acid sequence in SEQ ID NO: 2 as an antigen. When those two kindsof antibodies are used, it is possible to detect and differentiatenormal and A261del mutant proteins. The antibody of this inventionincludes all molecules which are able to bind to an epitope of an ALS2protein or other peptide of this invention, and all of Fab, F(ab′)₂, Fvfragments, etc. thereof. Such an antibody can be obtained from serumafter an animal is immunized using ALS2 derived protein or peptide as anantigen. Alternatively, the above expression vectors for eukaryoticcells may be introduced into muscle or skin of animals by injection orparticle gun and then serum is collected therefrom. Examples of animalsthat may be used are mouse, rat, rabbit, goat, chicken, etc. When Bcells collected from the spleen of an immunized animal are fused withmyeloma cells to produce a hybridoma, it is possible to producemonoclonal antibodies.

[0073] The diagnostic method of this invention is one in which an ALS2mutated gene or a transcription product of an ALS2 mutated gene isdetected whereby the risk of onset of ALS2 may be estimated.Particularly amenable are persons of known ALS2 families althoughdiagnosis is not limited thereto.

[0074] Genomic DNA of a person to be diagnosed may be subjected to a PCRamplification using any of the above-mentioned primer sets or otheroligonucleotides of this invention. The resulting DNA fragment may betreated with one or more restriction enzymes such as NarI, and theperson to be diagnosed where the DNA fragment is cleaved into fragmentsdifferent from cleaving product produced from a person not sufferingfrom ALS2 is indicative of a patient suffering from ALS2 or a personwith some risk of ALS2 in view of the presence of a mutation in the ALS2gene.

[0075] It is also possible to detect the ALS2 mutated genes by (forexample) an allele-specific oligonucleotide probe method, anoligonucleotide ligation assay method, a PCR-SSCP method, a PCR-CFLPmethod, a PCR-PHFA method, an invader method, an RCA (rolling circleamplification) method and a primer oligo base extension method.

[0076] In detecting transcription products of ALS2 mutated genes,diagnosis may be carried out by determining the sequence of mRNA of theperson to be diagnosed or cDNA thereof. It is also possible to carry outthe diagnosis in such a manner that an ALS2 gene of a person to bediagnosed or cDNA thereof is recombined with an expression vector,transfected to cells and the expression product thereof measured.

[0077] Expression products of normal and mutant ALS2 genes may beassessed by measurement of molecular weight. For example, the frameshift caused by deletion of one base in normal ALS2 gene whereupon themodified protein is changed to a low-molecular protein (SEQ ID NO: 3)comprising the first to the 46th amino acid residues of SEQ ID NO: 2 andthree amino acid residues (Pro-Ser-Glu) newly coded by the frame shiftresults in a product having a molecular weight easily comparable tonaturally occurring gene products of the normal ALS2 gene. Further,diagnosis may be also carried out by the above antibody provided by thisinvention in which the ALS2 modified protein reacts with an antibodyrecognizing (for example) the first to the 46th amino acid sequence inSEQ ID NO: 2 or a region comprising amino acids 43-49 of SEQ ID NO:3,but does not react with an antibody recognizing the 47th to the 1657thamino acid sequence region in SEQ ID NO: 2. Antibodies specific foramino acids 476 to 545 of SEQ ID NO:84 as compared to any of the aminoacids of SEQ ID NO:2 could be similarly used for diagnosis of theAG1548del. Diagnosis using antibodies may, for example, be carried outwith an ELIZA method.

[0078] The mouse ALS2 gene of this invention is a mouse genomic geneisolated as a homolog of the human ALS2 gene and which codes for a mouseALS2 protein comprising an amino acid sequence of SEQ ID NO: 5. Its CDNAhas a base sequence of SEQ ID NO: 4. This gene may be used for thepreparation of a “knock-out” mouse.

[0079] Such a “knock-out” mouse can be prepared by known gene targetingmethods (Science, 244: 1288-1292, 1989) or generally according to thefollowing example.

[0080] First, a DNA fragment of the mouse ALS2 gene including theinitiation codon of the gene is modified whereupon a defective DNAfragment which deletes expression of the ALS2 gene is obtained. Thisdefective DNA fragment is used for the preparation of a targeting vectorfor introduction of the modification into a mouse totipotent cell (EScell) according to known methods (such as the method described inScience, 244: 1288-1292, 1989). For example, genomic DNA comprising theALS2 gene is substituted or inserted with a resistant gene to acytotoxin to prepare a recombinant plasmid DNA possessing the defectivegene having a sequence homologous to the genomic DNA of the ALS2 gene atboth terminals (the targeting vector). It is also possible for theresistant gene to be connected to a sequence such as PGK1 promoter andPGK1 polyadenylation signal for controlling the expression. It ispreferred that the genomic DNA site of the ALS2CR6 gene which issubstituted with or inserted by resistant gene be a genomic DNA regioncontaining an exon region containing an initiation codon.

[0081] There are no particular limitations on such target vectors exceptthat it will have a sequence which is homologous to genomic DNA of theALS2 gene and a resistance sequence or other sequence useful for cellsorting (such as diphtheria toxin A gene and thymidine kinase gene ofherpes virus). A promoter and enhancer may be appropriately combined andused. The targeting vector is then introduced into an ES (embryonicstem) cell according to known methods (e.g. Nature, 292: 154-156, 1981).Such methods include electric pulse, a liposome and calcium phosphate.When recombination efficiency of the gene to be introduced is ofconcern, the electric pulse methods is preferred. DNA in each of the EScells into which gene is introduced is extracted and, by means of asouthern blot analysis or a PCR assay, cells are selected in which ahomologous gene recombination has taken place between the wild type ALS2gene existing on the chromosome and the introduced defective ALS2 genefragment resulting in placement of the defective gene fragment in thechromosome.

[0082] An ES cell having a defective gene prepared above may be injectedinto a blastocyst of a wild type animal and chimera-embryos obtainedwhich are transplanted to the uterus of a preliminary parent. Resultingprogeny are selected for the ALS2 defective gene and bred. Selection maybe carried out by checking the difference in the color of hair or byextraction of DNA from a part of the body (such as the tail end)followed by conducting a southern blot analysis, a PCR assay afterextraction of DNA, etc. As to the offspring obtained by a crossbreedingof animal of a wild type with a chimera animal where the ALS2 defectivegene is in the generative cells, a southern blot analysis, a PCR assayor the like may be carried out using the DNA extracted from a part ofthe body (such as the tail end) as a material to identify a heterozygoteinto which the ALS2 defective gene is introduced. A heterozygotepossessing the ALS2 defective gene which is stable in all generativecells and somatic cells may be bred to produce progeny in which the ALS2gene is completely “knocked-out”.

[0083] An animal prepared as such may be used for analysis of functionof ALS2 gene in onset of ALS2 and for screening of therapeutic drugs ordevelopment of therapeutic methods as an ALS2 model animal.

[0084] Methods and results of procedures carried out for cloning of theALS2 gene and for functional analysis thereof are shown.

[0085] 1. Methods

[0086] 1-1. ALS2 Family

[0087] Sixteen cases including 8 individuals suffering from the diseaseobtained from a Tunisian consanginous ALS2 family (literature 2) wereanalyzed. The characteristic of ALS2 is a progressive convulsion ofmuscles of the limbs and the face accompanied by distal myoatrophy ofthe hand and the foot. Age of onset is between 3 and 10 years age(literature 2). According to biopsy of nerves and muscles and also toelectromyography test, there was confirmed deletion of distal motorneuron (literature 2). When a gene type of the polymorphic DNA markerswas analyzed together with clinical test data, ALS2 was clearly anautosomal recessive inheritance.

[0088] 1-2. Transcription Map

[0089] Genome Data Base (GDB) (http://www.gdbwww.gdb.org) and UniGene(http://www.ncbi.nlm.nih.gov) of the Biotechnology Information Center(NCBI) which were open to the public for discriminating the sequence oftranscribed DNA mapped within an objective region were retrieved.Sequence of genomic DNA overlapped with the objective region of ALS2 wasretrieved from the “nr” or “htgs” data base of GenBank and utilized asthe object for the test when a BLAST retrieval to the dbEST data base isconducted. In order to isolate the transcript of a full length, therewere carried out RT-PCR, 5′-RACE and cDNA library screening. Inaddition, EST clone was purchased from Research Genetics and sequencingfor DNA was carried out for measuring the insertion of the whole clone.Sequence of double stranded DNA was determined by conducting a dideoxysequencing using a BigDye Terminator Cycle Sequencing Kit (ABI) and anAB1377DNA sequencer. All sequences of EST data, PCR products and DNAobtained from cDNA clone were determined and an estimated independenttranscription unit was established. Then each unit was mapped on aphysical map by a PCR method.

[0090] 1-3. Identification of Exon

[0091] In order to determine the constitution of intron and exon of thetranscription DNA, genomic DNA sequence data open to the public inGenBank data base was compared with the sequence of cDNA using aSequencer Version 3.0 (Gene Codes Corporation) program according to thedescriptions of BLAST (literature 28) and literatures (5 and 6).

[0092] 1-4. PCR

[0093] Exon and intron/exon boundaries were subjected to a PCRamplification. ExTaq polymerase (Takara) was used and a cycle of 95° C.for 15 seconds, 60° C. for 30 seconds and 72° C. for 30 seconds wasrepeated for 35 times whereby about 50 mg of genomic DNA were amplifiedby a PCR. In order to detect the deficient form of the transcriptionDNA, an RT-PCR was carried out. Total RNA from lymphocytes of fourpatients of a family of ALS2 and two carriers was isolated. Total RNAextracted from a healthy human brain was purchased from Clontech. AnRT-PCR was carried out using a SuperScript pre-amplification system(Gibco-BRL) according to the protocol of the manufacturer. Theoligonucleotide primer for such a PCR was designed using Primer 3.0(http://www-genome.wi.mit.edu). Table 1 lists the primers used foramplification of ALS2 (ALS2CR6). TABLE 1 Pro- SEQ SEQ duct ID ID Re-Exon I.D. (bp) Forward Primer NO Reverse Primer NO marks 1 CALS370ex01319 5′-GGAGAGACTGTGCTCCCAAG-3′ 10 5′-AGCCCTCCTAGCCAATAGC-3′ 11 2CALS370ex02 381 5′-TAAGCTTAGTGGGCAGGCTC-3′ 125′-TTCCCACTTAACAACCATCAAC-3′ 13 3 CALS370ex03 3395′-CCTAGTCATCCATGTGCTGG-3′ 6 5′-TCCCATACCTGACCTTCCAC-3′ 7 4CALS370ex04-1 424 5′-CCAATTTGGTTAAATCTATAGGGG-3′ 145′-GACAATGCCAGAGTGTGCTC-3′ 15 part of exon CALS370ex04-2 4355′-CCAGCCCTTTGTTAGCAGTC-3′ 16 5′-CTTCTTCCTGCCTGTCAAGG-3′ 17 part of exonCALS121 698 5′-TTGTACAATGCCTCCCTTCC-3′ 18 5′-AGCCCAACATGACACCTTC-3′ 19part of exon 5 CALS111ex002 490 5′-GATTGCTTGTTGCATAAGGG-3′ 205′-ATACAGCATGCGATGTCAGG-3′ 21 6 CALS120 322 5′-CTGGACTCCCACTCCTTCAC-3′22 5′-GCTAGAAGAGCCCAGATTTCC-3′ 23 7 CALS111ex004 4435′-TGACTTTGTGTGCCTGTGTG-3′ 24 5′-ATACCCTGGAAAATCTGGGG-3′ 25 8CALS111ex005 373 5′-TTTGCGCATTATCTCTGGTC-3′ 265′-GTACGTATGAAATTCCCCCG-3′ 27 9 CALS111ex006 3895′-TTCCGTCTTACTCCTGCACC-3′ 28 5′-GCCTTAGGATCCAATTCCTG-3′ 29 10CALS111ex007 467 5′-CAATGATGTACTGATGAACCAGC-3′ 305′-CCTGATGGTTTAATGGTGGG-3′ 31 11 CALS111ex008 3485′-GCACATGGCAACAGGTTAAG-3′ 32 5′-TCCTTGGCAGAATAACCGTG-3′ 33 12CALS111ex01 426 5′-CCCCTACCACTCCCTTTACC-3′ 345′-CCAGTGGCTAATAGTACCTGTCC-3′ 35 13 CALS111ex02 4805′-TGGATGCATGATTCATTTCC-3 36 5′-TCCTTGGCTTTCCAAATGTC-3′ 37 14CALS111ex03 462 5′-CTATCCTGGGGTCTCTGCTG-3′ 38 5′-TGCTATCGAAATGGTTGCTG-3′39 15 CALS111ex04 290 5′-AGCTACGACCAGCAAATTCC-3′ 405′-ATAGGGGTCCACCTTTCAGG-3′ 41 16 CALS111ex05 4545′-AAGGGGATATGGGCAGAGTC-3′ 42 5′-AAATGCTTGCTTGGTTTTGG-3′ 43 17CALS111ex06 340 5′-AAAGGGCATCTTCATTGCAC-3′ 44 5′-CACAAGAGGCAGAAAGAGCC-3′45 18 CALS111ex07 298 5′-AATGCTTGATGAATTGTTGCC-3′ 465′-ATGATCATCCTCACCCCAGG-3′ 47 19 CALS111ex08 3885′-TTGAAGATTTATGCCTGGGG-3′ 48 5′-TGAGGTCACACGGCTATCAG-3′ 49 20CALS111ex09 379 5′-GTGTAGTGGGGCTGATGTCC-3 50 5′-TGGCTATGCAAACATTCAGG-3′51 21 CALS111ex10 414 5′-AATGCAAAATACCACACATGG-3 525′-TCATTGGCTTAAACTGTGGG-3′ 53 22 CALS111ex11 4505′-CAACCTAGGGTTGATGCCTG-3′ 54 5′-CATCTTCGGAAAGCAAAACC-3′ 55 23CALS111ex12 205 5′-CTTTGGGGATATGACTGCGT-3′ 565′-GTAAAAGAATTTAGGGAGAAAAA-3′ 57 24 CALS111ex13 2605′-TTCCTCTAACCCCACATTTTATTC-3′ 58 5′-TGCTTTTAAAATATTAACCAGCTTTG-3′ 59 25CALS122exe01 320 5′-TCAGTCTTGGCAGTTTTGGTC-3′ 605′-CTGCTGTATGTTGAGCAGGTG-3′ 61 26 CALS122exe02 4555′-TGGATGCTCCACTTTGACTG-3′ 62 5′-TTAAGAACCCCCTTGAGTGC-3′ 63 27CALS122exe03 362 5′-TTCCTGGTCCCAAAATTGAC-3′ 645′-CAGGGTGAAACTACCCAAGC-3′ 65 28 CALS122exe04 2705′-TTTTATGCTTTTCAACCCCC-3′ 66 5′-ACACACTTTCTCGCTGGGAC-3′ 67 29CALS122exe05 394 5′-TGATCTGAGCACAAAGGCTG-3′ 685′-TAAACAGCGGTGGGTAGAGC-3′ 69 CALS122ex05 2855′-AATGCTCCTTTTCTCCCACTC-3′ 70 5′-TGCCAAATTTCCAATAATGC-3′ 71 30CALS122ex06 400 5′-TAATGGGGACAAGGAAGCC-3′ 72 5′-GCTGAGGCAAAACAAGCATC-3′73 31 CALS122ex07 375 5′-CCAAAGACCTGCACTCTGAC-3′ 745′-CTGGCTTGGCTCTCTCCTAC-3′ 75 32 CALS122ex08 3125′-AAAAAGCACGATCAAATGGC-3′ 76 5′-GGAAGAGCGTACTCCTGCTG-3′ 77 33CALS122ex09 393 5′-GCAGGAGTACGCTCTTCCAC-3′ 785′-GAACAAAATGTGCTCTAAAGGC-3′ 79 CALS24exY 486 5′-TCTTTTTCTCTCTGGGGCAG-3′80 5′-TGCCTTCTGTGTTTTACCCTG-3′ 81 34 CALS24exZ 4035′-GAAGGGAACAGGGAAAAGTG-3′ 82 5′-TTACCTCCCTTTCAATCCTCC3′ 83

[0094] 1-5. Analysis of Mutation

[0095] In order to detect the mutation of the DNA sequence at the exonor the intron/exon boundary, a DNA sequence of a PCR product of exon wasdetermined. The DNA sequence of a PCR product of exon was analyzed usingthe same oligonucleotide as a primer. The sequence in the data base opento the public was compared with the DNA sequence obtained from patients,carriers and healthy persons and changes in the nucleotide werediscriminated.

[0096] It was also confirmed that a new NarI site was formed (A261del)after the treatment with NarI by means of an RT-PCR amplification ofexons 2-4 or a PCR amplification of exon 3. As to the primers for exon3-PCR, there were used 5′-CCTAGTCATCCATGTGCTGG-3′ (SEQ ID NO: 6) and5′-TCCCATACCTGACCTTCCAC-3′ (SEQ ID NO: 7). As to the primers for theRT-PCR of exons 2-4, there were used 5′-CTTGATAGACTTTCTGTAAAGAAG-3′ (SEQID NO: 8) and 5′-GGCTACTTGGACAAATCTCCACTG-3′ (SEQ ID NO: 9). Decomposedproduct with NarI was separated by 1.5% agarose gel.

[0097] 1-6. Northern Blot Analysis

[0098] Northern (MTN) blot (Clontech) of many human adult tissues washybridized with exon 4 labelled with ³²P-dCTP of ALS2CR6 or humanβ-actin cDNA in a Perfect Hyb hybridizing solution (Toyobo). Themembrane was washed with 0.1×SSC containing 1% of SDS and subjected toan X-ray film (Bio-MAX, Kodak).

[0099] 1-7. MRNA In Situ Hybridization

[0100] Antisense and sense cRNA probes were prepared from two mouse cDNAclones m2-as and m2-s. Those mouse cDNA clones covered a part of mousemALS2CR6 cDNA (from the 1732nd to the 2685th bases of SEQ ID NO: 4; 954bp) and inserted into pCR2.1 (Invitrogen) in an opposite direction. Theprobes were prepared according to the protocol of the manufacturer(Roche Molecular Biochemicals) by an in vitro transcription reactionwhere digoxigenin-labelled UTP and T7 polymerase were mixed. Preparationof the sample and method for the in situ hybridization were inaccordance with the literature (29).

[0101] 1-8. Retrieval of the Data Base

[0102] Each of DNA and amino acid sequences was compared with the database of sequences of nucleotide and protein which were not overlappedeach other using BLASTN and BLASTP. Domain and motive of protein wereidentified by MOTIF servers of Genome Net Japan(http://www.genome.ad.jp), search launcher of BCM(http://www.hgse.hem.tmc.edu/Search.launcher) and CD search of NCBI(http://www.hcbi.nlm.nih.gov).

[0103] 2. Results

[0104] The inventors have prepared a physical map on the basis ofYAC/BAC/PAC of genomic region of 3 Mb covering a complete candidateregion to ALS2 (literatures 5 and 6). Sequences of EST and CDNA clonewere analyzed within a broad area and, at the same time, this physicalmap was used for the mapping of 43 independent transcription unitsincluding previously analyzed 18 genes (KIAA0005, CLK1, PP1L3, ORC2L,NDUFB3, CFLAR, CASP10, CASP8, FZD7, NOP5, UBL1, BMPR2, FLJ10881,LOC57404, AIP-1, CD28, CTLA4 and AILIM) and new 10 full-lengthtranscription products (ALS2CR1, ALS2CR2, ALS2CR3, ALS2CR4,ALS2CR5/MPP4, ALS2CR6, ALS2CR7, ALS2CR8, ALS2CR9 and ALS2CR12). Thosegenetic sequences were present in the locus of ALS2 (FIG. 1).

[0105] Juvenile ALS2 is rare and has a sign that, in teens and twenties,muscular convulsion of limbs, face and throat gradually expresses. SinceALS2 is recessively hereditary, it is predicted that this ALS2 diseasemay take place by a loss of a functional mutation. Big deletion ortranslocation in the ALS2 locus was investigated by a mapping of STS/ESTcontent on the basis of a PCR and a southern blot analysis but that wasnot detected. After that, small deletion or base substitution in exon orintron-exon boundary was investigated. In order to detect thosemutations, each gene was analyzed and an intron/exon boundary thereofwas determined. Until now, 395 exons have been identified from 42 genes.In order to amplify exon and flanking sequence thereof includingconsensus sequence to splicing donor and acceptor, 411 primers in totalwere designed and those primers were used to amplify the genomic DNA of10 normal control persons who were not related to 14 persons of the ALS2family (FIG. 2a) by PCR. Sequence of each of those PCR products wasdetermined whereby 77 sequence polymorphs in total of intron or exonwere identified. Among those 77 polymorphs, 8 mutations contained in 4different genes were related to ALS2 (Table 2). TABLE 2 Gene RegionNormal ALS2 NOP5 intron 2 tatctc(T)9aattct → (T)6 NOP5 intron 6gttttg(TTG)2ttttta → (TTG)3 ALS2CR6 intron 2 ggtaaAtcattt → G ALS2CR6exon 3 gcaggcAgccctc → A261 deletion* ALS2CR8 intron 6 gtcagtAttataa → GALS2CR9 exon 4 ctccagCatggac → T (3rd codon) ALS2CR9 intron 7ttgggaTtttttt → A ALS2CR9 intron 8 aaaataCggatat → T

[0106] Among those sequence mutations, one nucleotide deletion (A261del)noted in exon 3 of ALS2CR6 broke the reading frame and it is suggestedthat such a mutation mutates the protein. All of the suspicioushetero-conjugative carriers show a duplicated sequence pattern startingfrom the first nucleotide after the deficient part (FIG. 2b). Thisdeletion clearly moves together with an ALS2 expression type (FIG. 2c)and is not noted in 533 normal control individuals of various races(data not shown). In other mutations, one base substitution from C to Tin exon 4 of ALS2CR9 gene is included (C873T). However, this mutationcorresponds to the third codon and, therefore, it does not change theamino acid residue. In order to detect a splicing error which is madelatent or manifest by other sequence mutation, an RT-PCR was carried outusing total RNA extracted from lymphocytes of patients and healthycontrol persons but no sequence mutation of mRNA was detected (data notshown). Accordingly, the mutation related to ALS2 of intron or exonregion does not cause a splicing error. From those results, it has beenconfirmed that deletion of one base in exon 3 of ALS2CR6 (A261del;Table 1) is mutation concerning ALS2.

[0107] ALS2CR6 gene contains 33 introns and 34 exons and is present in agenomic DNA of 80.3 kb adjacent to a polymorphic DNA marker D2S2309(FIG. 1). Transcription polarity of the ALS2CR6 gene is in the directionof central body from telomere. An ALS2CR6 transcription product (mRNA)has a full length of 6394 bp (SEQ ID NO: 1) having a single open readingframe (ORF) with a length of 4974 nucleotides (124-5,097 nt) and codesfor a protein of 184 KDa comprising 1,657 amino acid residues.Polyadenylated estimated signal (AATAAA: 6,375-6,380 nt) and poly(A)region are clear. A short ALS2CR6 transcription product in a full lengthof 2,651 bp having 1,191 bp ORF coding for a 396 amino acid sequence wasidentified as well. This short variant splices a 5′-donor site afterexon 4 and, as a result, a stop codon is formed after 25 amino acidresidues of intron 4. Being correspondent to those results, 2transcription products of about 6.5 kb and 2.6 kb were identified inmany adult human tissues by a northern blot analysis (FIG. 3a). Exceptthe liver where short transcription products are mostly expressed, bothtranscription products showed the similar expression pattern. It hasbeen confirmed that a big transcription product of 6.5 kb is expressedin a slightly higher level than a transcription product of 2.6 kb and ismost abundantly expressed in the cerebellum. This gene has been alsoconfirmed to be expressed in cells of ALS2 patients (FIG. 3b).

[0108] Further, a mouse homolog of ALS2CR6 was isolated and namedmALS2CR6. A transcription product of mALS2CR6 is in a full length of6,349 bp (SEQ ID NO: 4) having one ORF of 4,956 bp (124-5,076 nt) andcodes for a protein of 183 kDa comprising 1651 amino acids (SEQ ID NO:5). The ORF as a whole is well reserved in a DNA level (87% same) and aprotein level (91% same; 94% similar; FIG. 4) between human being andmouse and it is suggested that ALS2CR6 gene is a gene which is wellreserved in mammals.

[0109] In order to check the localization property of expression ofmALS2CR6 transcription product in the brain and the spinal cord ofmouse, an in situ hybridization using riboprobe corresponding to a partof mALS2CR6 cDNA was carried out. The result was that, as shown in FIG.5, the mALS2CR6 transcription products were expressed in various levelsin nerve cells from the brain to the spinal cord, especially in neuronsof hippocampal and dentate gyrus, cerebellar Purkinje cells, neurons ofcerebral cortex and spinal cinerea including anterior horn cells. Inaddition, a significant expression was noted in neurons of olfactorybulb, basal nucleus and cranial nerve nucleus as well.

[0110] Human ALS2CR6 protein showed many interesting properties (FIG.6a). The first property is present in a region of A-terminal side ofALS2CR6 and it showed a high homology to RCC1 (regulatory factor forconcentrating the chromosome; literature 7) and RPGR (GTPase forpigmentary retinitis; literature 8)(FIG. 8). RCC1 and RPGR protein actsas a guanine nucleotide exchange factor (GEF) for GTPase like Ran. Thesecond property is that ALS2CR6 has a Db1-homologous (DH) domain and apleckstrin-homologous (PH) domain and both domains are typical domainsnoted in RhoGEF protein (literatures 9 and 10). In addition, VPS9 domainis noted in a C-terminal region as well. VPS9 domain is noted in manyGEF including Vps9 (literature 11) and Rabex-5 (literature 12) and eachis said to mediate the selection of vacuole protein and the phagocytictransportation. Two MORN motives comprising 14 amino acids (literature13) were noted as well. According to the recent study for junctophilincontaining an MORN motive, this motive is shown to contribute in bondingof plasma membrane (literature 13). It has been known that GEF isrelated to a GDP bonding form of GTPase and promotes the dissociation ofGDP and bonding of GTP whereby GTPase is activated. Since it has beenknown (literatures 18 and 19) that GEF plays an important role in manysignal transmission cascades (literature 14), neuron formation(literature 15), membrane transportation (literature 16) and formationof actin cell skeleton (literature 17), it is likely that ALS2CR6 actsas a regulatory factor/activator of Ran-related GTPase, regulates theformation of membrane and acts in a (membrane) transportation of cellsincluding neurons.

[0111] According to an RT-PCR analysis, a transcription product ofmutated ALS2CR6 gene is transcribed from chromosomes of the patient(FIG. 2c) and produces a modified protein comprising 49 amino acidshaving three new residues (Pro-Ser-Glu) at C-terminal (FIG. 6a). Sincethis modified protein has no functional domain corresponding to ALS2CR6protein, it seems to make the inherent function lost. Accordingly, theA261del mutation noted in this ALS2CR6 is related to the fact that ALS2is recessively hereditary.

[0112] A recent finding that ALS is related to defect for thetransportation of axon and the formation of cell skeleton (literatures20, 21 and 22) induces a hypothesis that ALS2CR6 gene corresponds toALS2 and that ALS2 is generated by the defect of membrane structure dueto lacking in a regulatory function of membrane structure Ran-relatedGTPase.

[0113] ALS2CR6 gene is the second ALS gene succeeding to thedetermination of role of copper-zinc superoxide desmutase (SDS-1) inALS. Mutation of SOD-1 is related to the form of tardive autosomaldominance (literature 23).

[0114] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of skill in the artin light of the teachings of this invention that changes andmodification may be made thereto without departing from the spirit orscope of the appended claims. All patents, patent applications andpublications referred to herein are hereby incorporated by reference.

References

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1 84 1 6394 DNA Homo sapiens CDS (124)..(5097) 1 ggacccactg ggttgccaagctcgcgccgg atgcggagcg cggtgctgcc ggtggagctt 60 caggtcttga tagactttctgtaaagaagg aatgatttgg tgatggagtg ttcccactga 120 ccg atg gac tca aag aagaga agc tca aca gag gca gaa gga tcc aag 168 Met Asp Ser Lys Lys Arg SerSer Thr Glu Ala Glu Gly Ser Lys 1 5 10 15 gaa aga ggc ctg gtc cat atctgg cag gca gga tcc ttt ccc ata aca 216 Glu Arg Gly Leu Val His Ile TrpGln Ala Gly Ser Phe Pro Ile Thr 20 25 30 cca gag aga ttg cca ggc tgg ggagga aag act gtt ttg cag gca gcc 264 Pro Glu Arg Leu Pro Gly Trp Gly GlyLys Thr Val Leu Gln Ala Ala 35 40 45 ctc gga gtg aaa cat gga gtt ctt ctgact gaa gat ggt gag gtc tac 312 Leu Gly Val Lys His Gly Val Leu Leu ThrGlu Asp Gly Glu Val Tyr 50 55 60 agc ttt ggg act ctt ccc tgg aga agt ggacca gtg gag att tgt cca 360 Ser Phe Gly Thr Leu Pro Trp Arg Ser Gly ProVal Glu Ile Cys Pro 65 70 75 agt agc ccc att cta gaa aat gcc ctg gtt gggcaa tat gtt att act 408 Ser Ser Pro Ile Leu Glu Asn Ala Leu Val Gly GlnTyr Val Ile Thr 80 85 90 95 gtg gca aca gga agc ttc cat agt gga gca gtgaca gac aat ggt gtc 456 Val Ala Thr Gly Ser Phe His Ser Gly Ala Val ThrAsp Asn Gly Val 100 105 110 gcg tac atg tgg gga gag aat tct gct ggc cagtgt gca gta gcc aac 504 Ala Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln CysAla Val Ala Asn 115 120 125 cag cag tat gtg ccg gaa cca aat cct gtc agcatt gct gat tct gag 552 Gln Gln Tyr Val Pro Glu Pro Asn Pro Val Ser IleAla Asp Ser Glu 130 135 140 gcc agc cct ttg tta gca gtc agg att tta cagttg gcg tgt ggc gag 600 Ala Ser Pro Leu Leu Ala Val Arg Ile Leu Gln LeuAla Cys Gly Glu 145 150 155 gag cac act ctg gca ttg tca ata agc aga gagatt tgg gca tgg ggt 648 Glu His Thr Leu Ala Leu Ser Ile Ser Arg Glu IleTrp Ala Trp Gly 160 165 170 175 acc ggt tgt cag ttg ggt ctc att acc actgcc ttc cca gtg aca aag 696 Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr AlaPhe Pro Val Thr Lys 180 185 190 ccg caa aag gta gaa cat ctt gct ggg cgagtg gtg ctt caa gtt gcc 744 Pro Gln Lys Val Glu His Leu Ala Gly Arg ValVal Leu Gln Val Ala 195 200 205 tgt ggt gct ttc cac agc tta gcc ctt gtacaa tgc ctc cct tcc cag 792 Cys Gly Ala Phe His Ser Leu Ala Leu Val GlnCys Leu Pro Ser Gln 210 215 220 gat ctg aag cca gtc cca gaa cga tgc aaccag tgc agc cag ctc ttg 840 Asp Leu Lys Pro Val Pro Glu Arg Cys Asn GlnCys Ser Gln Leu Leu 225 230 235 att act atg act gac aaa gaa gac cat gtgatt ata tca gac agt cat 888 Ile Thr Met Thr Asp Lys Glu Asp His Val IleIle Ser Asp Ser His 240 245 250 255 tgt tgc cca tta ggt gtg aca ctg acagaa tct cag gca gaa aac cat 936 Cys Cys Pro Leu Gly Val Thr Leu Thr GluSer Gln Ala Glu Asn His 260 265 270 gcc agc act gct ctc agc ccc tcc actgaa acc ctt gac agg cag gaa 984 Ala Ser Thr Ala Leu Ser Pro Ser Thr GluThr Leu Asp Arg Gln Glu 275 280 285 gaa gta ttt gag aac act ctt gta gcaaat gat cag tct gtt gct act 1032 Glu Val Phe Glu Asn Thr Leu Val Ala AsnAsp Gln Ser Val Ala Thr 290 295 300 gaa ctg aat gca gta agt gct cag atcaca agc agc gat gcc atg tcc 1080 Glu Leu Asn Ala Val Ser Ala Gln Ile ThrSer Ser Asp Ala Met Ser 305 310 315 tct caa caa aat gtc atg gga aca actgaa att tcc tct gcc aga aac 1128 Ser Gln Gln Asn Val Met Gly Thr Thr GluIle Ser Ser Ala Arg Asn 320 325 330 335 ata cca tca tac cct gac acc caagca gtc aat gaa tac cta cgg aaa 1176 Ile Pro Ser Tyr Pro Asp Thr Gln AlaVal Asn Glu Tyr Leu Arg Lys 340 345 350 ctg tca gat cat tca gta aga gaggac tca gag cat ggt gaa aag cca 1224 Leu Ser Asp His Ser Val Arg Glu AspSer Glu His Gly Glu Lys Pro 355 360 365 atg cca tct cag cct ctt tta gaagaa gca att cct aat ctc cac agc 1272 Met Pro Ser Gln Pro Leu Leu Glu GluAla Ile Pro Asn Leu His Ser 370 375 380 ccg cct acc aca agc acc tca gcccta aac agc ctg gtg gtc tct tgt 1320 Pro Pro Thr Thr Ser Thr Ser Ala LeuAsn Ser Leu Val Val Ser Cys 385 390 395 gca tct gct gtt ggt gtg aga gtggct gct act tat gaa gct ggt gcc 1368 Ala Ser Ala Val Gly Val Arg Val AlaAla Thr Tyr Glu Ala Gly Ala 400 405 410 415 ttg tca ctg aag aaa gtt atgaac ttt tat agt aca acc cct tgt gaa 1416 Leu Ser Leu Lys Lys Val Met AsnPhe Tyr Ser Thr Thr Pro Cys Glu 420 425 430 act gga gct cag gca ggc agtagt gcc att ggc ccc gaa ggt ttg aaa 1464 Thr Gly Ala Gln Ala Gly Ser SerAla Ile Gly Pro Glu Gly Leu Lys 435 440 445 gat agc agg gaa gaa cag gttaaa cag gaa tca atg caa gga aag aaa 1512 Asp Ser Arg Glu Glu Gln Val LysGln Glu Ser Met Gln Gly Lys Lys 450 455 460 agt tca agt ctt gtg gat atcaga gaa gaa gaa aca gag gga ggc agt 1560 Ser Ser Ser Leu Val Asp Ile ArgGlu Glu Glu Thr Glu Gly Gly Ser 465 470 475 cga aga ctc tcc ctc cct ggattg ttg tca caa gtt tcc ccc agg ctc 1608 Arg Arg Leu Ser Leu Pro Gly LeuLeu Ser Gln Val Ser Pro Arg Leu 480 485 490 495 tta aga aag gct gca cgggtg aaa acg agg aca gtg gtt ctg acc ccc 1656 Leu Arg Lys Ala Ala Arg ValLys Thr Arg Thr Val Val Leu Thr Pro 500 505 510 aca tac agt gga gaa gcagat gcg ctc ctg cct tct ctg aga aca gaa 1704 Thr Tyr Ser Gly Glu Ala AspAla Leu Leu Pro Ser Leu Arg Thr Glu 515 520 525 gtg tgg acc tgg ggg aaaggg aag gaa ggg cag ctg ggg cac ggc gat 1752 Val Trp Thr Trp Gly Lys GlyLys Glu Gly Gln Leu Gly His Gly Asp 530 535 540 gtt ctg cct agg ctt caaccg ttg tgt gta aaa tgt ctg gat ggc aaa 1800 Val Leu Pro Arg Leu Gln ProLeu Cys Val Lys Cys Leu Asp Gly Lys 545 550 555 gaa gta atc cat ctg gaggca ggt ggt tac cat tct ctt gca ctt act 1848 Glu Val Ile His Leu Glu AlaGly Gly Tyr His Ser Leu Ala Leu Thr 560 565 570 575 gcg aaa tcc cag gtttac tca tgg ggt agc aat acc ttt ggt caa ctt 1896 Ala Lys Ser Gln Val TyrSer Trp Gly Ser Asn Thr Phe Gly Gln Leu 580 585 590 ggg cat tcc gat tttcca aca aca gtt cct cgt ctt gca aag ata agc 1944 Gly His Ser Asp Phe ProThr Thr Val Pro Arg Leu Ala Lys Ile Ser 595 600 605 agt gaa aat gga gtctgg agc ata gct gca ggc agg gat tat tcc ctg 1992 Ser Glu Asn Gly Val TrpSer Ile Ala Ala Gly Arg Asp Tyr Ser Leu 610 615 620 ttt tta gtg gat acagaa gac ttc cag cct ggg tta tat tac agt ggc 2040 Phe Leu Val Asp Thr GluAsp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly 625 630 635 cga cag gac cct acagaa ggt gac aac ctt cca gag aat cac agt ggt 2088 Arg Gln Asp Pro Thr GluGly Asp Asn Leu Pro Glu Asn His Ser Gly 640 645 650 655 tct aag act ccagta ctt ctc tcc tgt agt aag ctt gga tat ata agc 2136 Ser Lys Thr Pro ValLeu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser 660 665 670 aga gtg aca gcagga aaa gat agc tat tta gcc ttg gtg gat aaa aac 2184 Arg Val Thr Ala GlyLys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn 675 680 685 att atg ggg tatatt gcc agt ctc cac gag tta gct act aca gaa aga 2232 Ile Met Gly Tyr IleAla Ser Leu His Glu Leu Ala Thr Thr Glu Arg 690 695 700 cga ttc tat tcaaaa cta agt gat atc aaa tct cag att ctc agg cct 2280 Arg Phe Tyr Ser LysLeu Ser Asp Ile Lys Ser Gln Ile Leu Arg Pro 705 710 715 ctt ctc agt ttagaa aat ttg ggc act aca act aca gtc cag ctg ttg 2328 Leu Leu Ser Leu GluAsn Leu Gly Thr Thr Thr Thr Val Gln Leu Leu 720 725 730 735 cag gag gtggct agc cga ttc agc aag ctg tgt tac ctc att ggt cag 2376 Gln Glu Val AlaSer Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln 740 745 750 cat gga gcctca ttg agc agc ttc ctt cat ggg gta aag gaa gcc agg 2424 His Gly Ala SerLeu Ser Ser Phe Leu His Gly Val Lys Glu Ala Arg 755 760 765 agt ttg gtcatc ctg aag cat tca agt ctc ttc ttg gat agt tat aca 2472 Ser Leu Val IleLeu Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr 770 775 780 gag tat tgcaca tct att aca aat ttc ctg gtt atg gga gga ttc cag 2520 Glu Tyr Cys ThrSer Ile Thr Asn Phe Leu Val Met Gly Gly Phe Gln 785 790 795 ctt ctt gctaag cct gcc att gat ttc cta aat aaa aac caa gag ctg 2568 Leu Leu Ala LysPro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu 800 805 810 815 ttg caagat ttg tca gaa gtg aat gac gaa aac act cag ttg atg gaa 2616 Leu Gln AspLeu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu 820 825 830 ata ctgaat act ttg ttt ttc ttg cca atc aga cga ctt cat aat tac 2664 Ile Leu AsnThr Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr 835 840 845 gca aaagtt ttg cta aag ctt gct act tgt ttt gaa gtg gca tct cca 2712 Ala Lys ValLeu Leu Lys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro 850 855 860 gaa tatcag aaa ctg cag gat tcc agt tct tgt tat gag tgt ctt gct 2760 Glu Tyr GlnLys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala 865 870 875 ctc catctc ggc agg aaa agg aag gaa gca gaa tac aca ctg ggc ttc 2808 Leu His LeuGly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe 880 885 890 895 tggaag acc ttc ccc gga aaa atg acg gat tcc ttg agg aag cca gag 2856 Trp LysThr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu 900 905 910 cgtcga ctg ctg tgt gag agt agt aac cga gcc ctg tct ctg cag cat 2904 Arg ArgLeu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His 915 920 925 gctggg agg ttt tcc gtg aat tgg ttc att ctc ttt aat gat gcc ctg 2952 Ala GlyArg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu 930 935 940 gtccat gcc cag ttc tcc acg cac cat gtt ttc cct ctg gcc acg ctg 3000 Val HisAla Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu 945 950 955 tgggca gag cca ctg tct gaa gaa gct ggt ggt gtg aat ggc tta aag 3048 Trp AlaGlu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn Gly Leu Lys 960 965 970 975ata act aca cct gag gag cag ttc act ctc att tca tct aca ccc cag 3096 IleThr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln 980 985 990gaa aag aca aag tgg cta cga gct ata agc caa gcc gta gat cag gct 3144 GluLys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala 995 10001005 ttg aga ggg atg tct gat ctc ccc cct tat gga agt ggt agc agt gtt3192 Leu Arg Gly Met Ser Asp Leu Pro Pro Tyr Gly Ser Gly Ser Ser Val1010 1015 1020 cag aga cag gaa cca ccc att tca cgc agt gcc aaa tat actttc tac 3240 Gln Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr PheTyr 1025 1030 1035 aag gat cct cgc cta aag gat gcc acc tat gat gga cgctgg ctt tca 3288 Lys Asp Pro Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg TrpLeu Ser 1040 1045 1050 1055 ggg aag cct cat ggc aga ggg gtt ttg aag tggcct gat gga aag atg 3336 Gly Lys Pro His Gly Arg Gly Val Leu Lys Trp ProAsp Gly Lys Met 1060 1065 1070 tat tct ggc atg ttc agg aat ggc ttg gaagat ggg tat gga gaa tac 3384 Tyr Ser Gly Met Phe Arg Asn Gly Leu Glu AspGly Tyr Gly Glu Tyr 1075 1080 1085 aga atc cca aac aag gca atg aac aaagaa gac cat tat gtg ggc cat 3432 Arg Ile Pro Asn Lys Ala Met Asn Lys GluAsp His Tyr Val Gly His 1090 1095 1100 tgg aaa gaa gga aaa atg tgc ggtcaa gga gtc tac agc tat gct tct 3480 Trp Lys Glu Gly Lys Met Cys Gly GlnGly Val Tyr Ser Tyr Ala Ser 1105 1110 1115 ggt gaa gta ttt gag ggc tgtttt caa gat aat atg cgt cat ggt cat 3528 Gly Glu Val Phe Glu Gly Cys PheGln Asp Asn Met Arg His Gly His 1120 1125 1130 1135 ggt ctt cta cga agtggg aaa ttg acg tcc tct tct cct agt atg ttc 3576 Gly Leu Leu Arg Ser GlyLys Leu Thr Ser Ser Ser Pro Ser Met Phe 1140 1145 1150 att ggc cag tgggta atg gat aag aaa gca gga tat ggt gtc ttt gat 3624 Ile Gly Gln Trp ValMet Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp 1155 1160 1165 gat atc actagg ggg gaa aag tat atg gga atg tgg caa gat gat gtg 3672 Asp Ile Thr ArgGly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val 1170 1175 1180 tgt caaggg aat ggt gtg gtg gtt acc cag ttt gga tta tac tac gag 3720 Cys Gln GlyAsn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu 1185 1190 1195 ggcaac ttt cac ctt aat aaa atg atg gga aat ggg gtt ttg ctt tcc 3768 Gly AsnPhe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser 1200 1205 12101215 gaa gat gat act atc tat gaa gga gaa ttt tca gat gac tgg act ctt3816 Glu Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu1220 1225 1230 agt gga aag gga aca ctg act atg cca aat gga gac tac attgaa ggt 3864 Ser Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp Tyr Ile GluGly 1235 1240 1245 tat ttt agt gga gaa tgg gga tct ggg ata aaa atc actgga acc tac 3912 Tyr Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr GlyThr Tyr 1250 1255 1260 ttc aaa cct agt cta tat gag agt gat aaa gac agacct aaa gtt ttc 3960 Phe Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Arg ProLys Val Phe 1265 1270 1275 agg aag cta gga aac ctg gca gtg cca gct gatgag aag tgg aaa gcg 4008 Arg Lys Leu Gly Asn Leu Ala Val Pro Ala Asp GluLys Trp Lys Ala 1280 1285 1290 1295 gtg ttt gac gaa tgt tgg cgc caa ctgggc tgt gag ggc cca ggc caa 4056 Val Phe Asp Glu Cys Trp Arg Gln Leu GlyCys Glu Gly Pro Gly Gln 1300 1305 1310 ggg gaa gtt tgg aaa gca tgg gacaat att gct gtg gcc ttg acc acc 4104 Gly Glu Val Trp Lys Ala Trp Asp AsnIle Ala Val Ala Leu Thr Thr 1315 1320 1325 agt cgg cgc cag cac aga gacagt cca gaa ata ttg agt cgt tca cag 4152 Ser Arg Arg Gln His Arg Asp SerPro Glu Ile Leu Ser Arg Ser Gln 1330 1335 1340 act cag aca cta gag agtttg gaa ttc att cca cag cat gtt ggt gcc 4200 Thr Gln Thr Leu Glu Ser LeuGlu Phe Ile Pro Gln His Val Gly Ala 1345 1350 1355 ttc tct gtg gag aaatat gat gac atc agg aaa tat tta ata aag gcc 4248 Phe Ser Val Glu Lys TyrAsp Asp Ile Arg Lys Tyr Leu Ile Lys Ala 1360 1365 1370 1375 tgt gac actcct ctg cac ccc ctg ggc agg ctt gtg gag aca ctg gtt 4296 Cys Asp Thr ProLeu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val 1380 1385 1390 gca gtgtat aga atg aca tac gtg ggc gta gga gcc aac cgc agg tta 4344 Ala Val TyrArg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu 1395 1400 1405 ttgcag gag gct gta aag gag att aag tcc tat ctt aag cga att ttc 4392 Leu GlnGlu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe 1410 1415 1420cag ctg gtg agg ttc tta ttt cct gag ctg cct gaa gaa ggc agc aca 4440 GlnLeu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr 1425 14301435 att cct ctc tct gct cct ctg cca acc gaa agg aag tct ttt tgc act4488 Ile Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr1440 1445 1450 1455 ggg aag tca gat tcc cga tct gaa tca cca gag cca ggttat gta gta 4536 Gly Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly TyrVal Val 1460 1465 1470 acg agt tct gga tta ttg ctt cct gtg ctg cta cctcgg ctc tac cca 4584 Thr Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro ArgLeu Tyr Pro 1475 1480 1485 ccg ctg ttt atg ctt tat gct ttg gat aat gatcgc gag gaa gac att 4632 Pro Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp ArgGlu Glu Asp Ile 1490 1495 1500 tac tgg gaa tgt gtc ctt cga cta aat aagcag cca gat att gct ctc 4680 Tyr Trp Glu Cys Val Leu Arg Leu Asn Lys GlnPro Asp Ile Ala Leu 1505 1510 1515 ctg ggc ttt ctt ggg gtg cag agg aaattt tgg cca gca acc ttg tca 4728 Leu Gly Phe Leu Gly Val Gln Arg Lys PheTrp Pro Ala Thr Leu Ser 1520 1525 1530 1535 atc ctt gga gag agt aaa aaggtt ttg cca acc acg aaa gat gct tgt 4776 Ile Leu Gly Glu Ser Lys Lys ValLeu Pro Thr Thr Lys Asp Ala Cys 1540 1545 1550 ttt gcc tca gca gta gaatgt ctg cag cag atc agc aca aca ttt acc 4824 Phe Ala Ser Ala Val Glu CysLeu Gln Gln Ile Ser Thr Thr Phe Thr 1555 1560 1565 cca tca gac aaa cttaag gtc atc cag cag act ttt gag gag atc tct 4872 Pro Ser Asp Lys Leu LysVal Ile Gln Gln Thr Phe Glu Glu Ile Ser 1570 1575 1580 cag agt gtc ctggcg tca ctc cac gaa gac ttc ttg tgg tcc atg gat 4920 Gln Ser Val Leu AlaSer Leu His Glu Asp Phe Leu Trp Ser Met Asp 1585 1590 1595 gac ttg tttcct gtt ttc tta tat gtg gtg cta cgg gcc agg att agg 4968 Asp Leu Phe ProVal Phe Leu Tyr Val Val Leu Arg Ala Arg Ile Arg 1600 1605 1610 1615 aattta ggc tct gag gta cac ctc att gag gat cta atg gac ccc tat 5016 Asn LeuGly Ser Glu Val His Leu Ile Glu Asp Leu Met Asp Pro Tyr 1620 1625 1630ctt cag cat ggg gaa cag ggt ata atg ttc acc acc ttg aag gca tgt 5064 LeuGln His Gly Glu Gln Gly Ile Met Phe Thr Thr Leu Lys Ala Cys 1635 16401645 tac tac cag att cag cgt gag aag ctt aac tag gctgcataac agcttgaaaa5117 Tyr Tyr Gln Ile Gln Arg Glu Lys Leu Asn 1650 1655 ctggattatctactacagag tgttataaca ccatctggag tcttcctgta gtggcaaaaa 5177 agaacagtgttgaaattgga aaggactttg tgttatttag gttgttagaa tgagccttac 5237 caataataagagccctgagc ccagaaaaaa ggactgtata gtttaaaggg aggattgaaa 5297 gggaggtaaaaaatcagatt agaccagttc ttggcctatg ataagttcca aaaataccat 5357 ttatctactatttgaaaaaa gaagaggata tcccttccta cagtaaaggg tatgtcagct 5417 acatgaagttgtaagaaaag cttccagtag agcttcttat attaaagaag ttgatggata 5477 tttttgaatttctggtttgc ctgaatccac ctgcagttac cccgatccgt ttgcaagaac 5537 cagatcgtacttgaaactat agtggccaca ctctgccttc ctgagtccct tccagtcatg 5597 tgtgcatcatgtctctttgc caagggaggg gagaaaggaa cttttaaact gcagttttaa 5657 ctttttctaagctgtttctt gatgggagag gttctgtgca aaactaccac attctgtccc 5717 caaaatgtggaatgcatcca aataggagtc ttctgcctct taacttaaaa gaacatagga 5777 attttgtttttggtttcttt atcatgctac agagagtgaa tacactggaa ttcagacacc 5837 gactctgagctgctaggaac ctcatttgtc catgtgcaaa cgctgtattc caaggcctgt 5897 gaatggcagcctgaggaagt tttgcatgca ggctgtgttt tcgagcagga ctaacaactg 5957 ggaaataagcaaaaaactgc atcgatcccc agcctggtgt tgttcttccc tatacttcac 6017 actgaactcaggatgggaag aaaaaggaaa caagctttgg ctttttccat ctcaaaagta 6077 ttgtggcacctcaacatttc agtgttttgc tttttaaaaa atgccctatt gtaagttgtt 6137 ggtttatactgtataagtaa cactagtagc tgttttgaat aacataggtg ctcttcctca 6197 tctcatctcctacaccgtgg tgagcataca gagtgtcctg atttgtgtta agtgactgag 6257 aagatgttaattacttttga aaaaggatca tggtttttgc tctactttat aatcaagaca 6317 agtgtttattaaaatactgt tttggaatgt tggctgtaat gtaacagcaa ttttcataat 6377 aaaaggcattcatcttt 6394 2 1657 PRT Homo sapiens 2 Met Asp Ser Lys Lys Arg Ser SerThr Glu Ala Glu Gly Ser Lys Glu 1 5 10 15 Arg Gly Leu Val His Ile TrpGln Ala Gly Ser Phe Pro Ile Thr Pro 20 25 30 Glu Arg Leu Pro Gly Trp GlyGly Lys Thr Val Leu Gln Ala Ala Leu 35 40 45 Gly Val Lys His Gly Val LeuLeu Thr Glu Asp Gly Glu Val Tyr Ser 50 55 60 Phe Gly Thr Leu Pro Trp ArgSer Gly Pro Val Glu Ile Cys Pro Ser 65 70 75 80 Ser Pro Ile Leu Glu AsnAla Leu Val Gly Gln Tyr Val Ile Thr Val 85 90 95 Ala Thr Gly Ser Phe HisSer Gly Ala Val Thr Asp Asn Gly Val Ala 100 105 110 Tyr Met Trp Gly GluAsn Ser Ala Gly Gln Cys Ala Val Ala Asn Gln 115 120 125 Gln Tyr Val ProGlu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu Ala 130 135 140 Ser Pro LeuLeu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu 145 150 155 160 HisThr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr 165 170 175Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro 180 185190 Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys 195200 205 Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp210 215 220 Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu LeuIle 225 230 235 240 Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser AspSer His Cys 245 250 255 Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln AlaGlu Asn His Ala 260 265 270 Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr LeuAsp Arg Gln Glu Glu 275 280 285 Val Phe Glu Asn Thr Leu Val Ala Asn AspGln Ser Val Ala Thr Glu 290 295 300 Leu Asn Ala Val Ser Ala Gln Ile ThrSer Ser Asp Ala Met Ser Ser 305 310 315 320 Gln Gln Asn Val Met Gly ThrThr Glu Ile Ser Ser Ala Arg Asn Ile 325 330 335 Pro Ser Tyr Pro Asp ThrGln Ala Val Asn Glu Tyr Leu Arg Lys Leu 340 345 350 Ser Asp His Ser ValArg Glu Asp Ser Glu His Gly Glu Lys Pro Met 355 360 365 Pro Ser Gln ProLeu Leu Glu Glu Ala Ile Pro Asn Leu His Ser Pro 370 375 380 Pro Thr ThrSer Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala 385 390 395 400 SerAla Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu 405 410 415Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr 420 425430 Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp 435440 445 Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser450 455 460 Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Glu Gly Gly SerArg 465 470 475 480 Arg Leu Ser Leu Pro Gly Leu Leu Ser Gln Val Ser ProArg Leu Leu 485 490 495 Arg Lys Ala Ala Arg Val Lys Thr Arg Thr Val ValLeu Thr Pro Thr 500 505 510 Tyr Ser Gly Glu Ala Asp Ala Leu Leu Pro SerLeu Arg Thr Glu Val 515 520 525 Trp Thr Trp Gly Lys Gly Lys Glu Gly GlnLeu Gly His Gly Asp Val 530 535 540 Leu Pro Arg Leu Gln Pro Leu Cys ValLys Cys Leu Asp Gly Lys Glu 545 550 555 560 Val Ile His Leu Glu Ala GlyGly Tyr His Ser Leu Ala Leu Thr Ala 565 570 575 Lys Ser Gln Val Tyr SerTrp Gly Ser Asn Thr Phe Gly Gln Leu Gly 580 585 590 His Ser Asp Phe ProThr Thr Val Pro Arg Leu Ala Lys Ile Ser Ser 595 600 605 Glu Asn Gly ValTrp Ser Ile Ala Ala Gly Arg Asp Tyr Ser Leu Phe 610 615 620 Leu Val AspThr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg 625 630 635 640 GlnAsp Pro Thr Glu Gly Asp Asn Leu Pro Glu Asn His Ser Gly Ser 645 650 655Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg 660 665670 Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile 675680 685 Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Thr Thr Glu Arg Arg690 695 700 Phe Tyr Ser Lys Leu Ser Asp Ile Lys Ser Gln Ile Leu Arg ProLeu 705 710 715 720 Leu Ser Leu Glu Asn Leu Gly Thr Thr Thr Thr Val GlnLeu Leu Gln 725 730 735 Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr LeuIle Gly Gln His 740 745 750 Gly Ala Ser Leu Ser Ser Phe Leu His Gly ValLys Glu Ala Arg Ser 755 760 765 Leu Val Ile Leu Lys His Ser Ser Leu PheLeu Asp Ser Tyr Thr Glu 770 775 780 Tyr Cys Thr Ser Ile Thr Asn Phe LeuVal Met Gly Gly Phe Gln Leu 785 790 795 800 Leu Ala Lys Pro Ala Ile AspPhe Leu Asn Lys Asn Gln Glu Leu Leu 805 810 815 Gln Asp Leu Ser Glu ValAsn Asp Glu Asn Thr Gln Leu Met Glu Ile 820 825 830 Leu Asn Thr Leu PhePhe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala 835 840 845 Lys Val Leu LeuLys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro Glu 850 855 860 Tyr Gln LysLeu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala Leu 865 870 875 880 HisLeu Gly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe Trp 885 890 895Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg 900 905910 Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala 915920 925 Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val930 935 940 His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr LeuTrp 945 950 955 960 Ala Glu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn GlyLeu Lys Ile 965 970 975 Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser SerThr Pro Gln Glu 980 985 990 Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln AlaVal Asp Gln Ala Leu 995 1000 1005 Arg Gly Met Ser Asp Leu Pro Pro TyrGly Ser Gly Ser Ser Val Gln 1010 1015 1020 Arg Gln Glu Pro Pro Ile SerArg Ser Ala Lys Tyr Thr Phe Tyr Lys 1025 1030 1035 1040 Asp Pro Arg LeuLys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly 1045 1050 1055 Lys ProHis Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr 1060 1065 1070Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg 10751080 1085 Ile Pro Asn Lys Ala Met Asn Lys Glu Asp His Tyr Val Gly HisTrp 1090 1095 1100 Lys Glu Gly Lys Met Cys Gly Gln Gly Val Tyr Ser TyrAla Ser Gly 1105 1110 1115 1120 Glu Val Phe Glu Gly Cys Phe Gln Asp AsnMet Arg His Gly His Gly 1125 1130 1135 Leu Leu Arg Ser Gly Lys Leu ThrSer Ser Ser Pro Ser Met Phe Ile 1140 1145 1150 Gly Gln Trp Val Met AspLys Lys Ala Gly Tyr Gly Val Phe Asp Asp 1155 1160 1165 Ile Thr Arg GlyGlu Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys 1170 1175 1180 Gln GlyAsn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly 1185 1190 11951200 Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu1205 1210 1215 Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp ThrLeu Ser 1220 1225 1230 Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp TyrIle Glu Gly Tyr 1235 1240 1245 Phe Ser Gly Glu Trp Gly Ser Gly Ile LysIle Thr Gly Thr Tyr Phe 1250 1255 1260 Lys Pro Ser Leu Tyr Glu Ser AspLys Asp Arg Pro Lys Val Phe Arg 1265 1270 1275 1280 Lys Leu Gly Asn LeuAla Val Pro Ala Asp Glu Lys Trp Lys Ala Val 1285 1290 1295 Phe Asp GluCys Trp Arg Gln Leu Gly Cys Glu Gly Pro Gly Gln Gly 1300 1305 1310 GluVal Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Ser 1315 13201325 Arg Arg Gln His Arg Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr1330 1335 1340 Gln Thr Leu Glu Ser Leu Glu Phe Ile Pro Gln His Val GlyAla Phe 1345 1350 1355 1360 Ser Val Glu Lys Tyr Asp Asp Ile Arg Lys TyrLeu Ile Lys Ala Cys 1365 1370 1375 Asp Thr Pro Leu His Pro Leu Gly ArgLeu Val Glu Thr Leu Val Ala 1380 1385 1390 Val Tyr Arg Met Thr Tyr ValGly Val Gly Ala Asn Arg Arg Leu Leu 1395 1400 1405 Gln Glu Ala Val LysGlu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln 1410 1415 1420 Leu Val ArgPhe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile 1425 1430 1435 1440Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr Gly 14451450 1455 Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val ValThr 1460 1465 1470 Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg LeuTyr Pro Pro 1475 1480 1485 Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp ArgGlu Glu Asp Ile Tyr 1490 1495 1500 Trp Glu Cys Val Leu Arg Leu Asn LysGln Pro Asp Ile Ala Leu Leu 1505 1510 1515 1520 Gly Phe Leu Gly Val GlnArg Lys Phe Trp Pro Ala Thr Leu Ser Ile 1525 1530 1535 Leu Gly Glu SerLys Lys Val Leu Pro Thr Thr Lys Asp Ala Cys Phe 1540 1545 1550 Ala SerAla Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro 1555 1560 1565Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln 15701575 1580 Ser Val Leu Ala Ser Leu His Glu Asp Phe Leu Trp Ser Met AspAsp 1585 1590 1595 1600 Leu Phe Pro Val Phe Leu Tyr Val Val Leu Arg AlaArg Ile Arg Asn 1605 1610 1615 Leu Gly Ser Glu Val His Leu Ile Glu AspLeu Met Asp Pro Tyr Leu 1620 1625 1630 Gln His Gly Glu Gln Gly Ile MetPhe Thr Thr Leu Lys Ala Cys Tyr 1635 1640 1645 Tyr Gln Ile Gln Arg GluLys Leu Asn 1650 1655 3 49 PRT Homo sapiens 3 Met Asp Ser Lys Lys ArgSer Ser Thr Glu Ala Glu Gly Ser Lys Glu 1 5 10 15 Arg Gly Leu Val HisIle Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro 20 25 30 Glu Arg Leu Pro GlyTrp Gly Gly Lys Thr Val Leu Gln Ala Pro Ser 35 40 45 Glu 4 6349 DNA Musmusculus CDS (124)..(5079) 4 ccacgcgtcc ggcggtgcag tcgggctcgc gccgggagaagagcgcggag ctgcgggagc 60 gtcaggtctt gagagagctt ttgctaatgg gatggtttggtgatggagta ctcctcctga 120 ccg atg gac tca aag aag aaa agc tca aca gaggca gaa gga tcc aaa 168 Met Asp Ser Lys Lys Lys Ser Ser Thr Glu Ala GluGly Ser Lys 1 5 10 15 gaa aga ggc cta gtc cat gtc tgg cag gca gga tccttt tct cta aca 216 Glu Arg Gly Leu Val His Val Trp Gln Ala Gly Ser PheSer Leu Thr 20 25 30 cca gag agg ttg cca ggc tgg ggt gga aag aca gtt cttcag gca gcc 264 Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu GlnAla Ala 35 40 45 ctt ggt gtg agg cat gga gtt ctt ctg act gaa gat ggt gaggtc tac 312 Leu Gly Val Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu ValTyr 50 55 60 agc ttt ggg act ctt ccc tgg aaa agt gaa tca gca gaa att tgtcca 360 Ser Phe Gly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro65 70 75 agc agc ccc ctt cta gaa agt gcc ctg gtt ggg cat cat gtt att act408 Ser Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr 8085 90 95 gtg gca aca ggg agc ttc cac agt gga gca gtg aca gag agc ggg gtg456 Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val 100105 110 gtg tac atg tgg gga gag aat gct gcc ggg cag tgt gcg gta gct aac504 Val Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn 115120 125 cag cag tat gtg ccg gag ccg agt cct gtc agc att tct gac tcg gag552 Gln Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu 130135 140 acc agc ccg tca tta gca gtt agg att ctg caa ttg gca tgt ggc gag600 Thr Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu 145150 155 gaa cac aca ctg gca ttg tca ctc agc aga gag atc tgg gca tgg ggc648 Glu His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp Ala Trp Gly 160165 170 175 acc ggc tgt cag ctg ggc ctc atc acc acc act ttc cca gtg acaaag 696 Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe Pro Val Thr Lys180 185 190 cca cag aag gtg gaa cac ctt gct gga cga gtg gtg ctc cag gtggcc 744 Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala195 200 205 tgc ggt gca ttc cac agc ctt gca ctt gtg cag tgc ctc cct cctcag 792 Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Pro Gln210 215 220 gat ctg aag cca gtc cca gag aga tgc aat cag tgc agc cag ctgctc 840 Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu225 230 235 atc acc atg aca gac aaa gag gac cat gtg ata ata tcg gac agccat 888 Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His240 245 250 255 tgc tgc cct tta ggt gtg aca ttg tcc gag tct caa gca gaaaag cat 936 Cys Cys Pro Leu Gly Val Thr Leu Ser Glu Ser Gln Ala Glu LysHis 260 265 270 gcc agc cct gct ccc agc cct cac cca gag gca ctg gat gagcag gga 984 Ala Ser Pro Ala Pro Ser Pro His Pro Glu Ala Leu Asp Glu GlnGly 275 280 285 gag gtg ttt gag aac acg gtg gta gaa gct gaa ctg aac atggga agc 1032 Glu Val Phe Glu Asn Thr Val Val Glu Ala Glu Leu Asn Met GlySer 290 295 300 agt cag acc aca agt ggc agt gcc att tcc acc cag cag aacatc gtg 1080 Ser Gln Thr Thr Ser Gly Ser Ala Ile Ser Thr Gln Gln Asn IleVal 305 310 315 gga aca gct gaa gtg tct tct gcc aga aca gct ccg tca taccca gac 1128 Gly Thr Ala Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr ProAsp 320 325 330 335 acc cat gcg gta act gca tac ctg cag aag ctg tca gagcat tcg atg 1176 Thr His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu HisSer Met 340 345 350 agg gag aac cat gag cct gga gaa aag cca ccc cag gtccag cct ctt 1224 Arg Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val GlnPro Leu 355 360 365 gta gaa gaa gca gtt cct gat ctt cac agt cca cca accaca agc acc 1272 Val Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr ThrSer Thr 370 375 380 tca gcc ctc aac agc ttg gtg gtc tcc tgt gca tct gctgtt ggt gtc 1320 Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala ValGly Val 385 390 395 aga gtg gct gcc acc tat gaa gct ggg gcc ttg tct ctcaag aaa gtt 1368 Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu LysLys Val 400 405 410 415 atg aac ttt tac agc act gcc ccc tgc gag acg gcagct cag tcg ggc 1416 Met Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala AlaGln Ser Gly 420 425 430 agt gcc tcc aca ggc cca gaa agt ctg aaa gat ctccga gaa gag cag 1464 Ser Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp Leu ArgGlu Glu Gln 435 440 445 gtg aaa cag gag tca ctg caa ggg aag aaa agc tcaagt ctc atg gac 1512 Val Lys Gln Glu Ser Leu Gln Gly Lys Lys Ser Ser SerLeu Met Asp 450 455 460 atc aga gag gaa gag tcg gag gga ggg agt cga agactc tcc ctc cca 1560 Ile Arg Glu Glu Glu Ser Glu Gly Gly Ser Arg Arg LeuSer Leu Pro 465 470 475 ggg ttg ttg tcg caa gtt tcc ccc agg ctc tta aggaag gct gcg agg 1608 Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg LysAla Ala Arg 480 485 490 495 gtg aaa act cgg aca gtg gtt ctg act ccc acatac agt gga gaa gca 1656 Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr TyrSer Gly Glu Ala 500 505 510 gat gcc ctt ctg cct tcc ctg agg aca gag gtgtgg acc tgg ggg aaa 1704 Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val TrpThr Trp Gly Lys 515 520 525 ggc aag gaa ggg cag cta ggg cac ggc gac gtcctg ccc agg ctt cag 1752 Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val LeuPro Arg Leu Gln 530 535 540 ccg ttg tgt gtc aag tgt ctg gat ggt aaa gaggta atc cac ctg gag 1800 Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu ValIle His Leu Glu 545 550 555 gcg ggc ggc tcc cac tcc ctc gca ctc act gcgaaa tct cag gtt tac 1848 Ala Gly Gly Ser His Ser Leu Ala Leu Thr Ala LysSer Gln Val Tyr 560 565 570 575 tca tgg ggc agt aat acc ttt ggt cag cttggg cat tct gag ttt cca 1896 Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu GlyHis Ser Glu Phe Pro 580 585 590 aca acg gtt cct cga ctc tca aag gtt agcagt gaa aat gga gtc tgg 1944 Thr Thr Val Pro Arg Leu Ser Lys Val Ser SerGlu Asn Gly Val Trp 595 600 605 agt gta gct gca ggc caa gat tat tcc ttgttt tta gtg gac acg gaa 1992 Ser Val Ala Ala Gly Gln Asp Tyr Ser Leu PheLeu Val Asp Thr Glu 610 615 620 gac ttc cag cct ggg ttg tat tac agt ggccga cag gac cgt gca gaa 2040 Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly ArgGln Asp Arg Ala Glu 625 630 635 ggt gat acc ctg cca gag aat ccc agt ggtaca aag act cca gta ctt 2088 Gly Asp Thr Leu Pro Glu Asn Pro Ser Gly ThrLys Thr Pro Val Leu 640 645 650 655 ctc tcc tgt agt aag ctt gga tac ataagc aga gta aca gca gga aaa 2136 Leu Ser Cys Ser Lys Leu Gly Tyr Ile SerArg Val Thr Ala Gly Lys 660 665 670 gat agc tat cta gcc ttg gtg gat aagaac atc atg gga tac atc gcc 2184 Asp Ser Tyr Leu Ala Leu Val Asp Lys AsnIle Met Gly Tyr Ile Ala 675 680 685 agt ctc cat gag ttg gct tct aca gaaaga cgg ttt tac tca aaa ctg 2232 Ser Leu His Glu Leu Ala Ser Thr Glu ArgArg Phe Tyr Ser Lys Leu 690 695 700 agc gaa atc aaa tca cag ata ctt aggcct ctt ctc agt tta gaa aat 2280 Ser Glu Ile Lys Ser Gln Ile Leu Arg ProLeu Leu Ser Leu Glu Asn 705 710 715 ttg ggc aca gtg acc act gtc cag ctgttg cag gaa gtt gcc agc cgg 2328 Leu Gly Thr Val Thr Thr Val Gln Leu LeuGln Glu Val Ala Ser Arg 720 725 730 735 ttc agc aag ttg tgt tac ctc attggg cag cat gga gcc tca cta agc 2376 Phe Ser Lys Leu Cys Tyr Leu Ile GlyGln His Gly Ala Ser Leu Ser 740 745 750 agc tac cta cag ggt atg aag gaagcc agc agc ctg gtc atc atg aag 2424 Ser Tyr Leu Gln Gly Met Lys Glu AlaSer Ser Leu Val Ile Met Lys 755 760 765 cac tca agt ctt ttc ctg gac agctac aca gag tac tgc aca tca gtt 2472 His Ser Ser Leu Phe Leu Asp Ser TyrThr Glu Tyr Cys Thr Ser Val 770 775 780 tca aat ttc ctg gtt atg gga ggattc cag ctt ctt gct aag cct gcc 2520 Ser Asn Phe Leu Val Met Gly Gly PheGln Leu Leu Ala Lys Pro Ala 785 790 795 att gat ttc cta aat aaa aac caagaa ctc ttg caa gat ttg tca gaa 2568 Ile Asp Phe Leu Asn Lys Asn Gln GluLeu Leu Gln Asp Leu Ser Glu 800 805 810 815 gtg aat gat gag aac act cagttg atg gaa atc ctg aac atg ctg ttt 2616 Val Asn Asp Glu Asn Thr Gln LeuMet Glu Ile Leu Asn Met Leu Phe 820 825 830 ttc ttg cca atc aga cga cttcat aat tat gca aaa gtt ttg cta aag 2664 Phe Leu Pro Ile Arg Arg Leu HisAsn Tyr Ala Lys Val Leu Leu Lys 835 840 845 ctt gcc act tgc ttt gaa gtgaca tct cca gag tat caa aag ctg cag 2712 Leu Ala Thr Cys Phe Glu Val ThrSer Pro Glu Tyr Gln Lys Leu Gln 850 855 860 gat tcc agt tct tgc tat gagtct ctt gct ctc cat ctt ggc aag aag 2760 Asp Ser Ser Ser Cys Tyr Glu SerLeu Ala Leu His Leu Gly Lys Lys 865 870 875 agg aag gaa gca gag tac acactg agc ttc tgg aag acc ttt cct ggg 2808 Arg Lys Glu Ala Glu Tyr Thr LeuSer Phe Trp Lys Thr Phe Pro Gly 880 885 890 895 aaa atg acg gat tcc ttgagg aag cca gag cgc cgg ctg ctg tgt gag 2856 Lys Met Thr Asp Ser Leu ArgLys Pro Glu Arg Arg Leu Leu Cys Glu 900 905 910 agc agt aac cga gcc ctctcc ctg cag cat gcc ggc agg ttt tct gtg 2904 Ser Ser Asn Arg Ala Leu SerLeu Gln His Ala Gly Arg Phe Ser Val 915 920 925 aat tgg ttc att ctc ttcaat gat gcc ctg gtc cat gct cag ttc tct 2952 Asn Trp Phe Ile Leu Phe AsnAsp Ala Leu Val His Ala Gln Phe Ser 930 935 940 aca cac cac gtg ttc cctttg gcc aca ctc tgg gca gag cca cta tct 3000 Thr His His Val Phe Pro LeuAla Thr Leu Trp Ala Glu Pro Leu Ser 945 950 955 gaa gaa gct ggt agc gtgaat ggc tta aag ata act aca cct gaa gaa 3048 Glu Glu Ala Gly Ser Val AsnGly Leu Lys Ile Thr Thr Pro Glu Glu 960 965 970 975 caa ttc aca ctc atttct tca aca ccc cag gaa aag acc aag tgg ctt 3096 Gln Phe Thr Leu Ile SerSer Thr Pro Gln Glu Lys Thr Lys Trp Leu 980 985 990 cgg gct att agc caagct gtg gat cag gct ttg agg ggg acg tcc gat 3144 Arg Ala Ile Ser Gln AlaVal Asp Gln Ala Leu Arg Gly Thr Ser Asp 995 1000 1005 ttc cca ctt tacgga ggc ggc agc agt gtt cag aga cag gaa cca ccc 3192 Phe Pro Leu Tyr GlyGly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro 1010 1015 1020 atc tca agaagt gcc aaa tac act ttc tac aag gat act cgc cta aag 3240 Ile Ser Arg SerAla Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys 1025 1030 1035 gat gccact tac gat ggg cgc tgg ctt tca ggg aag cct cat ggc agg 3288 Asp Ala ThrTyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg 1040 1045 1050 1055ggt gtt ctg aag tgg cct gat gga aag atg tac tct ggc atg ttc agg 3336 GlyVal Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg 1060 10651070 aat ggc ttg gaa gat gga tat ggt gaa tac aga atc cct aac aag gcc3384 Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg Ile Pro Asn Lys Ala1075 1080 1085 ctg aac aaa gaa gac cat tat gta ggc cat tgg aaa gag gggaaa atg 3432 Leu Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly LysMet 1090 1095 1100 tgt ggg caa gga gtc tac agc tat gcc tct ggt gaa gtgttt gaa ggc 3480 Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val PheGlu Gly 1105 1110 1115 tgc ttt caa gat aac atg cgc cat ggg cat ggt ctgctc cgg agt gga 3528 Cys Phe Gln Asp Asn Met Arg His Gly His Gly Leu LeuArg Ser Gly 1120 1125 1130 1135 aaa ctg act tct tct tct cct agc atg ttcatt ggc cag tgg gta atg 3576 Lys Leu Thr Ser Ser Ser Pro Ser Met Phe IleGly Gln Trp Val Met 1140 1145 1150 gat aag aaa gca gga tat ggc gtc tttgat gat atc acc agg gga gaa 3624 Asp Lys Lys Ala Gly Tyr Gly Val Phe AspAsp Ile Thr Arg Gly Glu 1155 1160 1165 aag tac atg gga atg tgg cag gatgat gtg tgc caa ggg aat ggg gta 3672 Lys Tyr Met Gly Met Trp Gln Asp AspVal Cys Gln Gly Asn Gly Val 1170 1175 1180 gta gtc acc cag ttt ggg ttatac tac gaa ggc aac ttc cac ctg aat 3720 Val Val Thr Gln Phe Gly Leu TyrTyr Glu Gly Asn Phe His Leu Asn 1185 1190 1195 aag atg atg gga aat ggggtt ttg ctt tct gaa gat gat acc atc tat 3768 Lys Met Met Gly Asn Gly ValLeu Leu Ser Glu Asp Asp Thr Ile Tyr 1200 1205 1210 1215 gaa gga gaa ttttcc gat gac tgg aca ctt agt gga aag gga acg ctg 3816 Glu Gly Glu Phe SerAsp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu 1220 1225 1230 act atg ccacat gga gat tat att gaa ggt tat ttt agt gga gaa tgg 3864 Thr Met Pro HisGly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp 1235 1240 1245 gga tctggg ata aaa atc act ggg acc tac ttc aaa cct agc ctg tat 3912 Gly Ser GlyIle Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr 1250 1255 1260 gagagc gat aag gac aag ccc aaa gcc ttc agg aag ctg ggg aac ctg 3960 Glu SerAsp Lys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu 1265 1270 1275gcc gtg gca gca gac gag aaa tgg aga gca gtg ttt gaa gaa tgc tgg 4008 AlaVal Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp 1280 12851290 1295 cac cag ctg ggc tgt gag agc cca ggc caa ggg gag gtt tgg aaagca 4056 His Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp Lys Ala1300 1305 1310 tgg gat aat att gct gtg gcc ttg acc acg aac cgt cgc cagcat aaa 4104 Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg Arg Gln HisLys 1315 1320 1325 gac agt cca gaa ata cta agc cgc tct cag act cag accctg gag agt 4152 Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr LeuGlu Ser 1330 1335 1340 ttg gag tac att ccc cag cac att ggc gcc ttc tctgtg gag aaa tat 4200 Leu Glu Tyr Ile Pro Gln His Ile Gly Ala Phe Ser ValGlu Lys Tyr 1345 1350 1355 gat gac atc aag aag tat tta ata aag gcc tgtgat act cct ctg cac 4248 Asp Asp Ile Lys Lys Tyr Leu Ile Lys Ala Cys AspThr Pro Leu His 1360 1365 1370 1375 cca ctg ggc agg ctt gtg gag acc ctggtt gcg gtg tat aga atg aca 4296 Pro Leu Gly Arg Leu Val Glu Thr Leu ValAla Val Tyr Arg Met Thr 1380 1385 1390 tat gtg ggt gta ggg gcc aac cgccgg tta ctg cag gaa gct gtg aag 4344 Tyr Val Gly Val Gly Ala Asn Arg ArgLeu Leu Gln Glu Ala Val Lys 1395 1400 1405 gag att aaa tct tat ctc aagagg att ttc cag ctt gtg agg ttc ttg 4392 Glu Ile Lys Ser Tyr Leu Lys ArgIle Phe Gln Leu Val Arg Phe Leu 1410 1415 1420 ttt cct gag ctt cct gaggag ggc agc aca att cct ctt tct gct cct 4440 Phe Pro Glu Leu Pro Glu GluGly Ser Thr Ile Pro Leu Ser Ala Pro 1425 1430 1435 ctg ccc act gga aggaga tcc ttc tgt act ggg aaa ttg gat tcc aga 4488 Leu Pro Thr Gly Arg ArgSer Phe Cys Thr Gly Lys Leu Asp Ser Arg 1440 1445 1450 1455 tcc gag tcacca gaa cca ggt tat gta gta aca agt tct ggc tta ctg 4536 Ser Glu Ser ProGlu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu 1460 1465 1470 ctt ccggtg ctg ctg ccg cgg ctc tac cca cct ctc ttc atg ctc tat 4584 Leu Pro ValLeu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr 1475 1480 1485 gccctg gat aat gac cga gag gaa gac att tac tgg gaa tgt gtg ctt 4632 Ala LeuAsp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu 1490 1495 1500cga cta aac aag cag cca gat att gct ctc ttg ggc ttc ctt gga gta 4680 ArgLeu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val 1505 15101515 cag aaa aaa ttc tgg cca gcc acc ttg tca atc ctt gga gag agt aaa4728 Gln Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys1520 1525 1530 1535 aag gtg ttg tca acc aca aag gat gct tgc ttt gca tctgca gta gaa 4776 Lys Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser AlaVal Glu 1540 1545 1550 tgc ctg cag cag atc agc aca aca ttt act cca tcagac aag ctt aaa 4824 Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser AspLys Leu Lys 1555 1560 1565 gtg atc cag cag acc ttt gaa gag atc tcc cagagt gtc ctt gca tcg 4872 Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln SerVal Leu Ala Ser 1570 1575 1580 ctg cag gag gac ttc ctc tgg tcc atg gatgac ttg ttc ccc gtc ttc 4920 Leu Gln Glu Asp Phe Leu Trp Ser Met Asp AspLeu Phe Pro Val Phe 1585 1590 1595 tta tac gtg gtg ctg cgg gcc agg attcgg aac ttg ggc tct gaa gtt 4968 Leu Tyr Val Val Leu Arg Ala Arg Ile ArgAsn Leu Gly Ser Glu Val 1600 1605 1610 1615 cac ctc att gag gat ctg atggac ccc ttt ctc cag cat ggg gaa caa 5016 His Leu Ile Glu Asp Leu Met AspPro Phe Leu Gln His Gly Glu Gln 1620 1625 1630 ggc atc atg ttc acc accttg aag gcc tgt tac ttc cag att cag cgg 5064 Gly Ile Met Phe Thr Thr LeuLys Ala Cys Tyr Phe Gln Ile Gln Arg 1635 1640 1645 gag aag ctt aac tagggcgcctgac agcttgagga ccggattatc tgctgcggag 5119 Glu Lys Leu Asn 1650gctacagcta tggcacaggc accgactgga ggctgatggg gcaaagaaca gtgttgaata 5179cagaatggac ttttgtgcta ttttggttgt aatttctgag ccttactaat aattagagcc 5239cagcatggaa aacatactgt atcattcaaa tggagactgg aaaaggagat agggatagag 5299tagagtcttt ggcctgtgct gagatccaca cacctactta gaaaaggaaa ctggttaccc 5359tttcctgtag tgaaagctct cagctccatg cagttccagg aaacctttcc aggaaagctg 5419cttagatgaa aagaagttga tgactgtgtt taagctcctg gtttgtctaa ttccatttgc 5479agttacccaa taccctttgg caaggagcag gttttacttg aaactgaagc agccatccct 5539tgccttccta gacctctcgc tcccaggcac aagtgcagca tgctactttg ctaggggtgg 5599gggtggggga gaagaagttt taaactgtag ttttaacctt ttgtaagccc ctttaccaag 5659gcatttgtgg tcagagagct cccacggggt gactatgaca tcctggtccc ctcgtggaat 5719gcatccacat aggatcttct gcctgctgac tgaaaagaac ataggaatac actggagtgc 5779aaacactgcc gtgccaagct gctccaaacc tcactgatcc gaggcccact gcctacccag 5839gaggcccact gcctacccag gaggcccgta agcttcttag cacaagcttt gtgtggagac 5899tgaagatctg cacatgtgag gaagcaggga gctacagtgg ccctcagccc agtctgcggg 5959tcttccctct acctcacact gaactcagaa gggaaggaag gagagacgca catgggattc 6019tcccacctca gaagtattgt gacagcaccg cataaccacg gtttgctctt ttacaagcag 6079cctcacaagt gtgggttgtg ggtgtgcgct ggagcagtgc cactcgtagc tgtttggata 6139ccacaggtgc tcttccgtct catctgctgt actcggaggc gagcgcagtg gcctgactca 6199tgggaaatga ctcagcaggc ggcaactact tttgaaaagg atcatgattt ccgagctact 6259ttataatcaa gacaagcatt tgttaacata ctgttttgga atgttggctg taatgtaaca 6319gcagttttca taataaatga cattcatctc 6349 5 1651 PRT Mus musculus 5 Met AspSer Lys Lys Lys Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu 1 5 10 15 ArgGly Leu Val His Val Trp Gln Ala Gly Ser Phe Ser Leu Thr Pro 20 25 30 GluArg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu 35 40 45 GlyVal Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser 50 55 60 PheGly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro Ser 65 70 75 80Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr Val 85 90 95Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val Val 100 105110 Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn Gln 115120 125 Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu Thr130 135 140 Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly GluGlu 145 150 155 160 His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp AlaTrp Gly Thr 165 170 175 Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe ProVal Thr Lys Pro 180 185 190 Gln Lys Val Glu His Leu Ala Gly Arg Val ValLeu Gln Val Ala Cys 195 200 205 Gly Ala Phe His Ser Leu Ala Leu Val GlnCys Leu Pro Pro Gln Asp 210 215 220 Leu Lys Pro Val Pro Glu Arg Cys AsnGln Cys Ser Gln Leu Leu Ile 225 230 235 240 Thr Met Thr Asp Lys Glu AspHis Val Ile Ile Ser Asp Ser His Cys 245 250 255 Cys Pro Leu Gly Val ThrLeu Ser Glu Ser Gln Ala Glu Lys His Ala 260 265 270 Ser Pro Ala Pro SerPro His Pro Glu Ala Leu Asp Glu Gln Gly Glu 275 280 285 Val Phe Glu AsnThr Val Val Glu Ala Glu Leu Asn Met Gly Ser Ser 290 295 300 Gln Thr ThrSer Gly Ser Ala Ile Ser Thr Gln Gln Asn Ile Val Gly 305 310 315 320 ThrAla Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr Pro Asp Thr 325 330 335His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu His Ser Met Arg 340 345350 Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val Gln Pro Leu Val 355360 365 Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr Thr Ser Thr Ser370 375 380 Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly ValArg 385 390 395 400 Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu LysLys Val Met 405 410 415 Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala AlaGln Ser Gly Ser 420 425 430 Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp LeuArg Glu Glu Gln Val 435 440 445 Lys Gln Glu Ser Leu Gln Gly Lys Lys SerSer Ser Leu Met Asp Ile 450 455 460 Arg Glu Glu Glu Ser Glu Gly Gly SerArg Arg Leu Ser Leu Pro Gly 465 470 475 480 Leu Leu Ser Gln Val Ser ProArg Leu Leu Arg Lys Ala Ala Arg Val 485 490 495 Lys Thr Arg Thr Val ValLeu Thr Pro Thr Tyr Ser Gly Glu Ala Asp 500 505 510 Ala Leu Leu Pro SerLeu Arg Thr Glu Val Trp Thr Trp Gly Lys Gly 515 520 525 Lys Glu Gly GlnLeu Gly His Gly Asp Val Leu Pro Arg Leu Gln Pro 530 535 540 Leu Cys ValLys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu Ala 545 550 555 560 GlyGly Ser His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr Ser 565 570 575Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly His Ser Glu Phe Pro Thr 580 585590 Thr Val Pro Arg Leu Ser Lys Val Ser Ser Glu Asn Gly Val Trp Ser 595600 605 Val Ala Ala Gly Gln Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu Asp610 615 620 Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Arg Ala GluGly 625 630 635 640 Asp Thr Leu Pro Glu Asn Pro Ser Gly Thr Lys Thr ProVal Leu Leu 645 650 655 Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val ThrAla Gly Lys Asp 660 665 670 Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile MetGly Tyr Ile Ala Ser 675 680 685 Leu His Glu Leu Ala Ser Thr Glu Arg ArgPhe Tyr Ser Lys Leu Ser 690 695 700 Glu Ile Lys Ser Gln Ile Leu Arg ProLeu Leu Ser Leu Glu Asn Leu 705 710 715 720 Gly Thr Val Thr Thr Val GlnLeu Leu Gln Glu Val Ala Ser Arg Phe 725 730 735 Ser Lys Leu Cys Tyr LeuIle Gly Gln His Gly Ala Ser Leu Ser Ser 740 745 750 Tyr Leu Gln Gly MetLys Glu Ala Ser Ser Leu Val Ile Met Lys His 755 760 765 Ser Ser Leu PheLeu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Val Ser 770 775 780 Asn Phe LeuVal Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala Ile 785 790 795 800 AspPhe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu Val 805 810 815Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Met Leu Phe Phe 820 825830 Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys Leu 835840 845 Ala Thr Cys Phe Glu Val Thr Ser Pro Glu Tyr Gln Lys Leu Gln Asp850 855 860 Ser Ser Ser Cys Tyr Glu Ser Leu Ala Leu His Leu Gly Lys LysArg 865 870 875 880 Lys Glu Ala Glu Tyr Thr Leu Ser Phe Trp Lys Thr PhePro Gly Lys 885 890 895 Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg LeuLeu Cys Glu Ser 900 905 910 Ser Asn Arg Ala Leu Ser Leu Gln His Ala GlyArg Phe Ser Val Asn 915 920 925 Trp Phe Ile Leu Phe Asn Asp Ala Leu ValHis Ala Gln Phe Ser Thr 930 935 940 His His Val Phe Pro Leu Ala Thr LeuTrp Ala Glu Pro Leu Ser Glu 945 950 955 960 Glu Ala Gly Ser Val Asn GlyLeu Lys Ile Thr Thr Pro Glu Glu Gln 965 970 975 Phe Thr Leu Ile Ser SerThr Pro Gln Glu Lys Thr Lys Trp Leu Arg 980 985 990 Ala Ile Ser Gln AlaVal Asp Gln Ala Leu Arg Gly Thr Ser Asp Phe 995 1000 1005 Pro Leu TyrGly Gly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro Ile 1010 1015 1020 SerArg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys Asp 1025 10301035 1040 Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly ArgGly 1045 1050 1055 Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly MetPhe Arg Asn 1060 1065 1070 Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg IlePro Asn Lys Ala Leu 1075 1080 1085 Asn Lys Glu Asp His Tyr Val Gly HisTrp Lys Glu Gly Lys Met Cys 1090 1095 1100 Gly Gln Gly Val Tyr Ser TyrAla Ser Gly Glu Val Phe Glu Gly Cys 1105 1110 1115 1120 Phe Gln Asp AsnMet Arg His Gly His Gly Leu Leu Arg Ser Gly Lys 1125 1130 1135 Leu ThrSer Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met Asp 1140 1145 1150Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu Lys 11551160 1165 Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly ValVal 1170 1175 1180 Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe HisLeu Asn Lys 1185 1190 1195 1200 Met Met Gly Asn Gly Val Leu Leu Ser GluAsp Asp Thr Ile Tyr Glu 1205 1210 1215 Gly Glu Phe Ser Asp Asp Trp ThrLeu Ser Gly Lys Gly Thr Leu Thr 1220 1225 1230 Met Pro His Gly Asp TyrIle Glu Gly Tyr Phe Ser Gly Glu Trp Gly 1235 1240 1245 Ser Gly Ile LysIle Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr Glu 1250 1255 1260 Ser AspLys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu Ala 1265 1270 12751280 Val Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp His1285 1290 1295 Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp LysAla Trp 1300 1305 1310 Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg ArgGln His Lys Asp 1315 1320 1325 Ser Pro Glu Ile Leu Ser Arg Ser Gln ThrGln Thr Leu Glu Ser Leu 1330 1335 1340 Glu Tyr Ile Pro Gln His Ile GlyAla Phe Ser Val Glu Lys Tyr Asp 1345 1350 1355 1360 Asp Ile Lys Lys TyrLeu Ile Lys Ala Cys Asp Thr Pro Leu His Pro 1365 1370 1375 Leu Gly ArgLeu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr Tyr 1380 1385 1390 ValGly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys Glu 1395 14001405 Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu Phe1410 1415 1420 Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser AlaPro Leu 1425 1430 1435 1440 Pro Thr Gly Arg Arg Ser Phe Cys Thr Gly LysLeu Asp Ser Arg Ser 1445 1450 1455 Glu Ser Pro Glu Pro Gly Tyr Val ValThr Ser Ser Gly Leu Leu Leu 1460 1465 1470 Pro Val Leu Leu Pro Arg LeuTyr Pro Pro Leu Phe Met Leu Tyr Ala 1475 1480 1485 Leu Asp Asn Asp ArgGlu Glu Asp Ile Tyr Trp Glu Cys Val Leu Arg 1490 1495 1500 Leu Asn LysGln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val Gln 1505 1510 1515 1520Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys Lys 15251530 1535 Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val GluCys 1540 1545 1550 Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp LysLeu Lys Val 1555 1560 1565 Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln SerVal Leu Ala Ser Leu 1570 1575 1580 Gln Glu Asp Phe Leu Trp Ser Met AspAsp Leu Phe Pro Val Phe Leu 1585 1590 1595 1600 Tyr Val Val Leu Arg AlaArg Ile Arg Asn Leu Gly Ser Glu Val His 1605 1610 1615 Leu Ile Glu AspLeu Met Asp Pro Phe Leu Gln His Gly Glu Gln Gly 1620 1625 1630 Ile MetPhe Thr Thr Leu Lys Ala Cys Tyr Phe Gln Ile Gln Arg Glu 1635 1640 1645Lys Leu Asn 1650 6 20 DNA Artificial Sequence Artificial SequenceSynthesized Oligonucleotide 6 cctagtcatc catgtgctgg 20 7 20 DNAArtificial Sequence Artificial Sequence Synthesized Oligonucleotide 7tcccatacct gaccttccac 20 8 24 DNA Artificial Sequence ArtificialSequence Synthesized Oligonucleotide 8 cttgatagac tttctgtaaa gaag 24 924 DNA Artificial Sequence Artificial Sequence SynthesizedOligonucleotide 9 ggctacttgg acaaatctcc actg 24 10 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide10 ggagagactg tgctcccaag 20 11 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 11 agccctcctt agccaatagc20 12 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 12 taagcttagt gggcaggctc 20 13 22 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 13 ttcccactta acaaccatca ac 22 14 24 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide14 ccaatttggt taaatctata gggg 24 15 20 DNA Artificial SequenceDescription of Artificial Sequence synthesized oligonucleotide 15gacaatgcca gagtgtgctc 20 16 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 16 ccagcccttt gttagcagtc20 17 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 17 cttcttcctg cctgtcaagg 20 18 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 18 ttgtacaatg cctcccttcc 20 19 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide19 agcccaacat gacacctttc 20 20 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 20 gattgcttgt tgcataaggg20 21 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 21 atacagcatg cgatgtcagg 20 22 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 22 ctggactccc actccttcac 20 23 21 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide23 gctagaagag cccagatttc c 21 24 20 DNA Artificial Sequence Descriptionof Artificial Sequence synthesized oligonucleotide 24 tgactttgtgtgcctgtgtg 20 25 20 DNA Artificial Sequence Description of ArtificialSequence synthesized oligonucleotide 25 ataccctgga aaatctgggg 20 26 20DNA Artificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 26 tttgcgcatt atctctggtc 20 27 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide27 gtacgtatga aattcccccg 20 28 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 28 ttccgtctta ctcctgcacc20 29 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 29 gccttaggat ccaattcctg 20 30 23 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 30 caatgatgta ctgatgaacc agc 23 31 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide31 cctgatggtt taatggtggg 20 32 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 32 gcacatggca acaggttaag20 33 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 33 tccttggcag aataaccctg 20 34 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 34 cccctaccac tccctttacc 20 35 23 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide35 ccagtggcta atagtacctg tcc 23 36 20 DNA Artificial SequenceDescription of Artificial Sequence synthesized oligonucleotide 36tggatgcatg attcatttcc 20 37 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 37 tccttggctt tccaaatgtc20 38 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 38 ctatcctggg gtctctgctg 20 39 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 39 tgctatcgaa atggttgctg 20 40 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide40 agctacgacc agcaaattcc 20 41 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 41 ataggggtcc acctttcagg20 42 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 42 aaggggatat gggcagagtc 20 43 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 43 aaatgcttgc ttggttttgg 20 44 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide44 aaagggcatc ttcattgcac 20 45 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 45 cacaagaggc agaaagagcc20 46 21 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 46 aatgcttgat gaattgttgc c 21 47 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 47 atgatcatcc tcaccccagg 20 48 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide48 ttgaagattt atgcctgggg 20 49 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 49 tgaggtcaca cggctatcag20 50 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 50 gtgtagtggg gctgatgtcc 20 51 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 51 tggctatgca aacattcagg 20 52 21 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide52 aatgcaaaat accacacatg g 21 53 20 DNA Artificial Sequence Descriptionof Artificial Sequence synthesized oligonucleotide 53 tcattggcttaaactgtggg 20 54 20 DNA Artificial Sequence Description of ArtificialSequence synthesized oligonucleotide 54 caacctaggg ttgatgcctg 20 55 20DNA Artificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 55 catcttcgga aagcaaaacc 20 56 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide56 ctttggggat atgactgcgt 20 57 26 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 57 gtaaaagaat ttattagggagaaaaa 26 58 24 DNA Artificial Sequence Description of ArtificialSequence synthesized oligonucleotide 58 ttcctctaac cccacatttt attc 24 5926 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 59 tgcttttaaa atattaacca gctttg 26 60 21 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 60 tcagtcttgg cagttttggt c 21 61 21 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide61 ctgctgtatg ttgagcaggt g 21 62 20 DNA Artificial Sequence Descriptionof Artificial Sequence synthesized oligonucleotide 62 tggatgctccactttgactg 20 63 20 DNA Artificial Sequence Description of ArtificialSequence synthesized oligonucleotide 63 ttaagaaccc ccttgagtgc 20 64 20DNA Artificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 64 ttcctggtcc caaaattgac 20 65 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide65 cagggtgaaa ctacccaagc 20 66 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 66 ttttatgctt ttcaaccccc20 67 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 67 acacactttc tcgctgggac 20 68 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 68 tgatctgagc acaaaggctg 20 69 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide69 taaacagcgg tgggtagagc 20 70 21 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 70 aatgctcctt ttctcccactc 21 71 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 71 tgccaaattt ccaataatgc 20 72 19 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 72 taatggggac aaggaagcc 19 73 20 DNA Artificial SequenceDescription of Artificial Sequence synthesized oligonucleotide 73gctgaggcaa aacaagcatc 20 74 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 74 ccaaagacct gcactctgac20 75 20 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 75 ctggcttggc tctctcctac 20 76 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 76 aaaaagcacg atcaaatggc 20 77 20 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide77 ggaagagcgt actcctgctg 20 78 20 DNA Artificial Sequence Description ofArtificial Sequence synthesized oligonucleotide 78 gcaggagtac gctcttccac20 79 22 DNA Artificial Sequence Description of Artificial Sequencesynthesized oligonucleotide 79 gaacaaaatg tgctctaaag gc 22 80 20 DNAArtificial Sequence Description of Artificial Sequence synthesizedoligonucleotide 80 tctttttctc tctggggcag 20 81 21 DNA ArtificialSequence Description of Artificial Sequence synthesized oligonucleotide81 tgccttctgt gttttaccct g 21 82 20 DNA Artificial Sequence Descriptionof Artificial Sequence synthesized oligonucleotide 82 gaagggaacagggaaaagtg 20 83 21 DNA Artificial Sequence Description of ArtificialSequence synthesized oligonucleotide 83 ttacctccct ttcaatcctc c 21 84545 PRT Homo sapiens 84 Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala GluGly Ser Lys Glu 1 5 10 15 Arg Gly Leu Val His Ile Trp Gln Ala Gly SerPhe Pro Ile Thr Pro 20 25 30 Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr ValLeu Gln Ala Ala Leu 35 40 45 Gly Val Lys His Gly Val Leu Leu Thr Glu AspGly Glu Val Tyr Ser 50 55 60 Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro ValGlu Ile Cys Pro Ser 65 70 75 80 Ser Pro Ile Leu Glu Asn Ala Leu Val GlyGln Tyr Val Ile Thr Val 85 90 95 Ala Thr Gly Ser Phe His Ser Gly Ala ValThr Asp Asn Gly Val Ala 100 105 110 Tyr Met Trp Gly Glu Asn Ser Ala GlyGln Cys Ala Val Ala Asn Gln 115 120 125 Gln Tyr Val Pro Glu Pro Asn ProVal Ser Ile Ala Asp Ser Glu Ala 130 135 140 Ser Pro Leu Leu Ala Val ArgIle Leu Gln Leu Ala Cys Gly Glu Glu 145 150 155 160 His Thr Leu Ala LeuSer Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr 165 170 175 Gly Cys Gln LeuGly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro 180 185 190 Gln Lys ValGlu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys 195 200 205 Gly AlaPhe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp 210 215 220 LeuLys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile 225 230 235240 Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys 245250 255 Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His Ala260 265 270 Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln GluGlu 275 280 285 Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val AlaThr Glu 290 295 300 Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp AlaMet Ser Ser 305 310 315 320 Gln Gln Asn Val Met Gly Thr Thr Glu Ile SerSer Ala Arg Asn Ile 325 330 335 Pro Ser Tyr Pro Asp Thr Gln Ala Val AsnGlu Tyr Leu Arg Lys Leu 340 345 350 Ser Asp His Ser Val Arg Glu Asp SerGlu His Gly Glu Lys Pro Met 355 360 365 Pro Ser Gln Pro Leu Leu Glu GluAla Ile Pro Asn Leu His Ser Pro 370 375 380 Pro Thr Thr Ser Thr Ser AlaLeu Asn Ser Leu Val Val Ser Cys Ala 385 390 395 400 Ser Ala Val Gly ValArg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu 405 410 415 Ser Leu Lys LysVal Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr 420 425 430 Gly Ala GlnAla Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp 435 440 445 Ser ArgGlu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser 450 455 460 SerSer Leu Val Asp Ile Arg Glu Glu Glu Thr Gly Arg Gln Ser Lys 465 470 475480 Thr Leu Pro Pro Trp Ile Val Val Thr Ser Phe Pro Gln Ala Leu Lys 485490 495 Lys Gly Cys Thr Gly Glu Asn Glu Asp Ser Gly Ser Asp Pro His Ile500 505 510 Gln Trp Arg Ser Arg Cys Ala Pro Ala Phe Ser Glu Asn Arg SerVal 515 520 525 Asp Leu Gly Glu Arg Glu Gly Arg Ala Ala Gly Ala Arg ArgCys Ser 530 535 540 Ala 545

What is claimed is:
 1. An isolated nucleic acid that codes for a peptidehaving at least 75% identity to all of an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:5; SEQID NO:84; and, amino acids 372-1657 of SEQ ID NO:2.
 2. The nucleic acidof claim 1 that codes for a peptide having about 80% or more sequenceidentity to the selected sequence.
 3. The nucleic acid of claim 1 thatcodes for a peptide having about 85% or more sequence identity to theselected sequence.
 4. The nucleic acid of claim 1 that codes for apeptide having about 90% or more sequence identity to the selectedsequence.
 5. The nucleic acid of claim 1 that codes a peptide havingabout 95% or more sequence identity to the selected sequence.
 6. Thenucleic acid of any one of claims 1-5, wherein the selected sequence isSEQ ID NO:2.
 7. The nucleic acid of claim 1, wherein the selectedsequence is SEQ ID NO:3.
 8. The nucleic acid of claim 1, wherein theselected sequence is SEQ ID NO:5.
 9. The nucleic acid of claim 1,wherein the selected sequence is SEQ ID NO:84.
 10. An isolated nucleicacid consisting essentially of a nucleotide sequence having at least 75%identity to all of a nucleotide sequence or a complementary sequencethereof, selected from the group consisting of: SEQ ID NO:1; SEQ IDNO:4; nucleotides 124-5094 of SEQ ID NO:1; nucleotides 1225-5094 of SEQID NO:1; and, nucleotides 124-5076 of SEQ ID NO:4.
 11. The nucleic acidof claim 10 having about 80% or more sequence identity to the selectedsequence or complementary sequence thereof.
 12. The nucleic acid ofclaim 10 having about 85% or more sequence identity to the selectedsequence or complementary sequence thereof.
 13. The nucleic acid ofclaim 10 having about 90% or more sequence identity to the selectedsequence or complementary sequence thereof.
 14. The nucleic acid ofclaim 10 having about 95% or more sequence identity to the selectedsequence or complementary sequence thereof.
 15. The nucleic acid of anyone of claims 10-14, wherein the selected sequence is SEQ ID NO:1. 16.The nucleic acid of any one of claims 10-14, wherein the selectedsequence is SEQ ID NO:4.
 17. The nucleic acid of any one of claims10-14, wherein the selected sequence is nucleotides 124-5094 of SEQ IDNO:
 1. 18. The nucleic acid of any one of claims 10-14, wherein theselected sequence is amino acids 124-5076 of SEQ ID NO:4.
 19. Theisolated nucleic acid of any one of claims 1-18 joined to a secondnucleic acid, wherein the second nucleic acid is not naturallyassociated with the isolated nucleic acid.
 20. A recombinant vectorcomprising a nucleic acid according to any one of claims 1-19.
 21. Acell comprising a nucleic acid of claim 19 or a vector of claim
 20. 22.An oligonucleotide of 6 to 75 nucleotides, wherein the oligonucleotidehybridizes to a nucleic acid according to any one of claims 1-18 or acomplementary sequence thereof, under stringent conditions.
 23. Theoligonucleotide of claim 22 of about 10 to about 40 nucleotides.
 24. Theoligonucleotide of claim 22 of about 15 to about 30 nucleotides.
 25. Theoligonucleotide of claim 22 of about 15 to about 25 nucleotides.
 26. Theoligonucleotide of any one of claims 22-25 capable of hybridizing understringent conditions to a nucleic acid encoding a peptide consisting ofthe sequence of SEQ ID NO:3 or the complementary nucleic acid sequencethereof, but not to a nucleic acid encoding a peptide consisting of thesequence of SEQ ID NO:2 or the complementary nucleic acid sequencethereof.
 27. The oligonucleotide of any one of claims 22-25 capable ofhybridizing under stringent conditions to a nucleic acid encoding apeptide consisting of the sequence of SEQ ID NO:84 or the complementarynucleic acid sequence thereof, but not to a nucleic acid encoding apeptide consisting of the sequence of SEQ ID NO:2 or the complementarynucleic acid sequence thereof.
 28. The oligonucleotide of any one ofclaims 22-27 joined to a label.
 29. A kit comprising two or moredifferent oligonucleotides according to any one of claims 22-27 for usein nucleic acid amplification.
 30. An isolated peptide comprising asequence of amino acids coded by a nucleic acid according to any one ofclaims 1-19 or a recombinant vector according to claim
 20. 31. A peptideconsisting essentially of a sequence of at least 5 contiguous aminoacids from a sequence selected from the group consisting of: amino acids146 of SEQ ID NO:2; amino acids 47-1657 of SEQ ID NO:2; SEQ ID NO:3;amino acids 43-49 of SEQ ID NO:3; SEQ ID NO:84; and amino acids 476 to545 of SEQ ID NO:84.
 32. A peptide comprising at least 5 contiguousamino acids from amino acids 4349 of SEQ ID NO:3 or amino acids 476 to545 of SEQ ID NO:84.
 33. An antibody which binds a peptide according toany one of claims 30-32.
 34. The antibody of claim 33 prepared by usinga peptide according to any one of claims 30-32 as an antigen.
 35. Anon-human mammal comprising a mutated gene, wherein the gene but for themutation would encode a protein having at least 75% sequence identity toall of SEQ ID NO:2 or SEQ ID NO:5.
 36. The mammal of claim 35, whereinthe protein has at least 85% sequence identity to all of SEQ ID NO:1 orSEQ ID NO:2.
 37. The mammal of claim 35 or 36, wherein the mutated genedoes not express a protein having biological activity.
 38. The mammal ofclaim 35, 36, or 37, wherein the mutated gene is incapable of expressionof a protein.
 39. The mammal of any one of claims 35-38, wherein themammal is a rodent.
 40. The mammal of claim 39, wherein the rodent is amouse.
 41. A method for the diagnosis of amyotrophic lateral sclerosistype 2 in a patient, comprising detecting the presence of a mutation ina gene that encodes a protein having at least 75% sequence identity toSEQ ID NO:2.
 42. The method of claim 41, wherein the protein has atleast about 90% sequence identity to SEQ ID NO:2.
 43. The method ofclaim 41, wherein the protein has at least about 95% sequence identityto SEQ ID NO:2.
 44. The method of claim 41, wherein the protein has atleast about 97% sequence identity to SEQ ID NO:2.
 45. The method ofclaim 41, wherein the protein has essentially the sequence of SEQ IDNO:2 but for the presence of the mutation.
 46. The method of any one ofclaims 41-45, comprising detecting the presence of the mutation in abiological sample from the patient.
 47. The method of any one of claims41-46, wherein the detecting comprises comparing a sequence of the gene,a RNA transcript of the gene, and a cDNA made from the RNA transcript,or a protein expressed by the gene from a human patient, to SEQ ID NO:1,wherein a difference in sequence is indicative of mutation.
 48. Themethod of claim 46, comprising contacting nucleic acids obtained fromthe biological sample or cDNA made from said nucleic acids, with one ormore oligonucleotides according to any one of claims 22 to
 28. 49. Themethod of claim 46, comprising detecting whether the one or moreoligonucleotides hybridize to said nucleic acids or cDNA, understringent conditions.
 50. The method of claim 46, comprisingamplification of nucleic acids or cDNA to which two or more of saidoligonucleotides hybridize, and determining the presence of an amplifiedproduct.
 51. A method for the diagnosis of amyotrophic lateral sclerosistype 2, comprising detecting the presence or absence of a protein havingat least 85% sequence identity to all of SEQ ID NO:2 in a patient.
 52. Amethod for the diagnosis of amyotrophic lateral sclerosis type 2,comprising detecting the presence or absence of a protein having atleast 95% sequence identity to all of SEQ ID NO:2 in a patient.
 53. Themethod of claim 51, wherein the detecting comprises determining whethera protein having at least 85% sequence identity to all of SEQ ID NO:2 ispresent in a biological sample from the patient.
 54. The method of claim52, wherein the detecting comprises determining whether a protein havingat least 95% sequence identity to all of SEQ ID NO:2 is present in abiological sample from the patient.
 55. A method for the diagnosis ofamyotrophic lateral sclerosis type 2, comprising detecting the presenceor absence of a protein having at least 85% sequence identity to all ofSEQ ID NO:3 or SEQ ID NO:84 in a biological sample from the patient. 56.A method for the diagnosis of amyotrophic lateral sclerosis type 2,comprising detecting the presence or absence of a protein having atleast 95% sequence identity to all of SEQ ED NO:3 or SEQ ID NO:84 in abiological sample from the patient.
 57. The method of any one of claims51-56, comprising contacting an antibody according to any one of claims33 or 34 with a biological sample from the patient and determiningwhether the antibody binds to protein in the sample.
 58. A method oftreatment of amyotrophic lateral sclerosis type 2, comprisingadministering a peptide, a nucleic acid, or a pharmaceutical compositioncomprising the peptide or nucleic acid to a patient in need thereof,wherein the peptide comprises an amino acid sequence having at least 90%identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acidcodes for said peptide.
 59. A method of treatment of amyotrophic lateralsclerosis type 2, comprising administering a peptide, a nucleic acid, ora pharmaceutical composition comprising the peptide or nucleic acid to apatient in need thereof, wherein the peptide comprises an amino acidsequence having at least 95% identity to SEQ ID NO:2 or a fragmentthereof, and the nucleic acid codes for said peptide.
 60. A method oftreatment of amyotrophic lateral sclerosis type 2, comprisingadministering a composition to a patient in need thereof, wherein thecomposition mimics the biological activity of the peptide of SEQ ID NO.2.
 61. The use of a peptide or a nucleic acid for preparation of amedicament for treatment of amyotrophic lateral sclerosis type 2,wherein the peptide comprises an amino acid sequence having at least 90%identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acidcodes for said peptide.
 62. The use of a peptide or a nucleic acid forpreparation of a medicament for treatment of amyotrophic lateralsclerosis type 2, wherein the peptide comprises an amino acid sequencehaving at least 95% identity to SEQ ID. NO:2 or a fragment thereof, andthe nucleic acid codes for said peptide.